Sulfonamide and carbamide derivatives of 6(5H)phenanthridinones and their uses

ABSTRACT

This invention relates to compounds, pharmaceutical compositions, and methods of using the disclosed compounds for inhibiting PARP.

[0001] The present application claims benefit of the filing of U.S.Provisional Application No. 60/205,259, filed May 19, 2000, the entirecontents of which is incorporated by reference herein.

[0002] The present invention relates to inhibitors of the nuclear enzymepoly(adenosine 5′-diphospho-ribose) polymerase [“poly(ADP-ribose)polymerase” or “PARP”, which is also referred to as ADPRT (NAD:protein(ADP-ribosyl transferase (polymersing)) and PARS (poly(ADP-ribose)synthetase) and provides compounds and compositions containing thedisclosed compounds. Moreover, the present invention provides methods ofusing the disclosed PARP inhibitors to prevent and/or treat tissuedamage resulting from cell damage or death due to necrosis or apoptosis;neural tissue damage resulting from, for example, ischemia andreperfusion injury, such as cerebral ischemic stroke, head trauma orspinal cord injury; neurological disorders and neurodegenerativediseases, such as, for example, Parkinson's or Alzheimer's diseases andmultiple sclerosis; to prevent or treat vascular stroke; to treat orprevent cardiovascular disorders, such as, for example, myocardialinfarction; to treat other conditions and/or disorders such as, forexample, age-related muscular degeneration, AIDS and other immunesenescence diseases, inflammation, arthritis, gout, atherosclerosis,cachexia, cancer, degenerative diseases of skeletal muscle involvingreplicative senescence, hyperglycemia, diabetes (such as diabetesmellitus), inflammatory bowel disorders (such as colitis and Crohn'sdisease), acute pancreatitis, mucositis, hemorrhagic shock, splanchnicartery occlusion shock, multiple organ failure (such as involving any ofthe kidney, liver, renal, pulmonary, retinal, pancreatic and/or skeletalmuscle systems), acute autoimmune thyroiditis, muscular dystrophy,osteoarthritis, osteoporosis, chronic and acute pain (such asneuropathic pain), renal failure, retinal ischemia, septic shock (suchas endotoxic shock), local and/or remote endothelial cell dysfunction(such are recognized by endo-dependent relaxant responses andup-regulation of adhesion molecules), inflammation and skin aging; toextend the lifespan and proliferative capacity of cells, such as, forexample, as general mediators in the generation of oxidants,proinflammatory mediators and/or cytokines, and general mediators ofleukocyte infiltration, calcium ion overload, phospholipid peroxidation,impaired nitric oxide metabolism and/or reduced ATP production; to altergene expression of senescent cells; or to radiosensitize hypoxic tumorcells.

[0003] PARP (EC 2.4.2.30), also known as PARS (for poly(ADP-ribose)synthetase), or ADPRT (for NAD:protein (ADP-ribosyl) transferase(polymerising)) is a major nuclear protein of 116 kDa. It is mainlypresent in almost all eukaryotes. The enzyme synthesizespoly(ADP-ribose), a branched polymer that can consist of over 200ADP-ribose units from NAD. The protein acceptors of poly(ADP-ribose) aredirectly or indirectly involved in maintaining DNA integrity. Theyinclude histones, topoisomerases, DNA and RNA polymerases, DNA ligases,and Ca²+- and Mg²+-dependent endonucleases. PARP protein is expressed ata high level in many tissues, most notably in the immune system, heart,brain and germ-line cells. Under normal physiological conditions, thereis minimal PARP activity. However, DNA damage causes an immediateactivation of PARP by up to 500-fold. Among the many functionsattributed to PARP is its major role in facilitating DNA repair byADP-ribosylation and therefore co-ordinating a number of DNA repairproteins. As a result of PARP activation, NAD levels significantlydecline. While many endogenous and exogenous agents have been shown todamage DNA and activate PARP, peroxynitrite, formed from a combinationof nitric oxide (NO) and superoxide, appears to be a main perpetratorresponsible for various reported disease conditions in vivo, e.g.,during shock, stroke and inflammation

[0004] Extensive PARP activation leads to severe depletion of NAD incells suffering from massive DNA damage. The short life ofpoly(ADP-ribose) (half-life <1 min) results in a rapid turnover rate.Once poly(ADP-ribose) is formed, it is quickly degraded by theconstitutively active poly(ADP-ribose) glycohydrolase (PARG), togetherwith phosphodiesterase and (ADP-ribose) protein lyase. PARP and PARGform a cycle that converts a large amount of NAD to ADP-ribose. In lessthan an hour, over-stimulation of PARP can cause a drop of NAD and ATPto less than 20% of the normal level. Such a scenario is especiallydetrimental during ischaemia when deprivation of oxygen has alreadydrastically compromised cellular energy output. Subsequent free radicalproduction during reperfusion is assumed to be a major cause of tissuedamage. Part of the ATP drop, which is typical in many organs duringischaemia and reperfusion, could be linked to NAD depletion due topoly(ADP-ribose) turnover. Thus, PARP or PARG inhibition is expected topreserve the cellular energy level to potentiate the survival ofischaemic tissues after insult.

[0005] Poly(ADP-ribose) synthesis is also involved in the inducedexpression of a number of genes essential for inflammatory response.PARP inhibitors suppress production of inducible nitric oxide synthase(iNOS) in macrophages, P-type selectin and intercellular adhesionmolecule-1 (ICAM-1) in endothelial cells. Such activity underlies thestrong anti-inflammation effects exhibited by PARP inhibitors. PARPinhibition is able to reduce necrosis by preventing translocation andinfiltration of neutrophils to the injured tissues. (Zhang, J. “PARPinhibition: a novel approach to treat ischaemia/reperfusion andinflammation-related injuries”, Chapter 10 in Emerging Drugs (1999) 4:209-221 Ashley Publications Ltd., and references cited therein.)

[0006] PARP production is activated by damaged DNA fragments which, onceactivated, catalyzes the attachment of up to 100 ADP-ribose units to avariety of nuclear proteins, including histones and PARP itself. Duringmajor cellular stresses the extensive activation of PARP can rapidlylead to cell damage or death through depletion of energy stores. As fourmolecules of ATP are consumed for every molecule of NAD (the source ofADP-ribose and PARP substrate) regenerated, NAD is depleted by massivePARP activation and, in the efforts to re-synthesize NAD, ATP may alsobe depleted.

[0007] It has been reported that PARP activation plays a key role inboth NMDA- and NO-induced neurotoxicity. This has been demonstrated incortical cultures and in hippocampal slices wherein prevention oftoxicity is directly correlated to PARP inhibition potency (Zhang etal., “Nitric Oxide Activation of Poly(ADP-Ribose) Synthetase inNeurotoxicity”, Science, 263:687-89 (1994) and Wallis et al.,“Neuroprotection Against Nitric Oxide Injury with Inhibitors ofADP-Ribosylation”, NeuroReport, 5:3, 245-48 (1993)). The potential roleof PARP inhibitors in treating neurodegenerative diseases and headtrauma has thus been recognized even if the exact mechanism of actionhas not yet been elucidated (Endres et al., “Ischemic Brain Injury isMediated by the Activation of Poly(ADP-Ribose)Polymerase”, J. Cereb.Blood Flow Metabol., 17:1143-51 (1997) and Wallis et al., “TraumaticNeuroprotection with Inhibitors of Nitric Oxide and ADP-Ribosylation,Brain Res., 710:169-77 (1996)).

[0008] Similarly, it has been demonstrated that single injections ofPARP inhibitors have reduced the infarct size caused by ischemia andreperfilsion of the heart or skeletal muscle in rabbits. In thesestudies, a single injection of 3-amino-benzamide (10 mg/kg), either oneminute before occlusion or one minute before reperfusion, caused similarreductions in infarct size in the heart (32-42%) whilel,5-dihydroxyisoquinoline (1 mg/kg), another PARP inhibitor, reducedinfarct size by a comparable degree (38-48%). Thiemermann et al.,“Inhibition of the Activity of Poly(ADP Ribose) Synthetase ReducesIschemia-Reperfusion Injury in the Heart and Skeletal Muscle”, Proc.Natl. Acad. Sci. USA, 94:679-83 (1997). These results make it reasonableto suspect that PARP inhibitors could salvage previously ischemic heartor skeletal muscle tissue.

[0009] PARP activation can also be used as a measure of damage followingneurotoxic insults following over-exposure to any of glutamate (via NMDAreceptor stimulation), reactive oxygen intermediates, amyloid β-protein,N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or its activemetabolite N-methyl-4-phenylpyridine (MPP⁺), which participate inpathological conditions such as stroke, Alzheimer's disease andParkinson's disease. Zhang et al., “Poly(ADP-Ribose) SynthetaseActivation: An Early Indicator of Neurotoxic DNA Damage”, J. Neurochem.,65:3, 1411-14 (1995). Other studies have continued to explore the roleof PARP activation in cerebellar granule cells in vitro and in MPTPneurotoxicity. Cosi et al., “Poly(ADP-Ribose) Polymerase (PARP)Revisited. A New Role for an Old Enzyme: PARP Involvement inNeurodegeneration and PARP Inhibitors as Possible NeuroprotectiveAgents”, Ann. N. Y. Acad. Sci., 825:366-79 (1997); and Cosi et al.,“Poly(ADP-Ribose) Polymerase Inhibitors Protect Against MPTP-inducedDepletions of Striatal Dopamine and Cortical Noradrenaline in C57B1/6Mice”, Brain Res., 729:264-69 (1996). Excessive neural exposure toglutamate, which serves as the predominate central nervous systemneurotransmitter and acts upon the N-methyl-D-aspartate (NMDA) receptorsand other subtype receptors, most often occurs as a result of stroke orother neurodegenerative processes. Oxygen deprived neurons releaseglutamate in great quantities during ischemic brain insult such asduring a stroke or heart attack. This excess release of glutamate inturn causes over-stimulation (excitotoxicity) of N-methyl-D-aspartate(NMDA), AMPA, Kainate and MGR receptors, which open ion channels andpermit uncontrolled ion flow (e.g., Ca²⁺ and Na⁺ into the cells and K⁺out of the cells) leading to overstimulation of the neurons. Theover-stimulated neurons secrete more glutamate, creating a feedback loopor domino effect which ultimately results in cell damage or death viathe production of proteases, lipases and free radicals. Excessiveactivation of glutamate receptors has been implicated in variousneurological diseases and conditions including epilepsy, stroke,Alzheimer's disease, Parkinson's disease, Amyotrophic Lateral Sclerosis(ALS), Huntington's disease, schizophrenia, chronic pain, ischemia andneuronal loss following hypoxia, hypoglycemia, ischemia, trauma, andnervous insult. Glutamate exposure and stimulation has also beenimplicated as a basis for compulsive disorders, particularly drugdependence. Evidence includes findings in many animal species, as wellas in cerebral cortical cultures treated with glutamate or NMDA, thatglutamate receptor antagonists (i.e., compounds which block glutamatefrom binding to or activating its receptor) block neural damagefollowing vascular stroke. Dawson et al., “Protection of the Brain fromIschemia”, Cerebrovascular Disease, 319-25 (H. Hunt Batjer ed., 1997).Attempts to prevent excitotoxicity by blocking NMDA, AMPA, Kainate andMGR receptors have proven difficult because each receptor has multiplesites to which glutamate may bind and hence finding an effective mix ofantagonists or universal antagonist to prevent binding of glutamate toall of the receptor and allow testing of this theory, has beendifficult. Moreover, many of the compositions that are effective inblocking the receptors are also toxic to animals. As such, there ispresently no known effective treatment for glutamate abnormalities.

[0010] The stimulation of NMDA receptors by glutamate, for example,activates the enzyme neuronal nitric oxide synthase (nNOS), leading tothe formation of nitric oxide (NO), which also mediates neurotoxicity.NMDA neurotoxicity may be prevented by treatment with nitric oxidesynthase (NOS) inhibitors or through targeted genetic disruption of nNOSin vitro. Dawson et al., “Nitric Oxide Mediates Glutamate Neurotoxicityin Primary Cortical Cultures”, Proc. Natl. Acad. Sci. USA, 88:6368-71(1991); and Dawson et al., “Mechanisms of Nitric Oxide-mediatedNeurotoxicity in Primary Brain Cultures”, J. Neurosci., 13:6, 2651-61(1993), Dawson et al., “Resistance to Neurotoxicity in Cortical Culturesfrom Neuronal Nitric Oxide Synthase-Deficient Mice”, J. Neurosci., 16:8,2479-87 (1996), ladecola, “Bright and Dark Sides of Nitric Oxide inIschemic Brain Injury”, Trends Neurosci., 20:3, 132-39 (1997), Huang etal., “Effects of Cerebral Ischemia in Mice Deficient in Neuronal NitricOxide Synthase”, Science, 265:1883-85 (1994), Beckman et al.,“Pathological Implications of Nitric Oxide, Superoxide and PeroxynitriteFormation”, Biochem. Soc. Trans., 21:330-34 (1993), and Szabó et al.,“DNA Strand Breakage, Activation of Poly(ADP-Ribose) Synthetase, andCellular Energy Depletion are Involved in the Cytotoxicity inMacrophages and Smooth Muscle Cells Exposed to Peroxynitrite”, Proc.Natl. Acad. Sci. USA, 93:1753-58 (1996).

[0011] It is also known that PARP inhibitors, such as 3-amino benzamide,affect DNA repair generally in response, for example, to hydrogenperoxide or gamma-radiation. Cristovao et al., “Effect of aPoly(ADP-Ribose) Polymerase Inhibitor on DNA Breakage and CytotoxicityInduced by Hydrogen Peroxide and γ-Radiation,” Terato., Carcino., andMuta., 16:219-27 (1996). Specifically, Cristovao et al. observed aPARP-dependent recovery of DNA strand breaks in leukocytes treated withhydrogen peroxide.

[0012] PARP inhibitors have been reported to be effective inradiosensitizing hypoxic tumor cells and effective in preventing tumorcells from recovering from potentially lethal damage of DNA afterradiation therapy, presumably by their ability to prevent DNA repair.U.S. Pat. Nos. 5,032,617; 5,215,738; and 5,041,653.

[0013] Evidence also exists that PARP inhibitors are useful for treatinginflammatory bowel disorders, such as colitis. Salzman et al., “Role ofPeroxynitrite and Poly(ADP-Ribose)Synthase Activation ExperimentalColitis,” Japanese J. Pharm., 75, Supp. I:15 (1997). Specifically,Colitis was induced in rats by intraluminal administration of the haptentrinitrobenzene sulfonic acid in 50% ethanol. Treated rats received3-aminobenzamide, a specific inhibitor of PARP activity. Inhibition ofPARP activity reduced the inflammatory response and restored themorphology and the energetic status of the distal colon. See also,Southan et al., “Spontaneous Rearrangement of Aminoalkylithioureas intoMercaptoalkylguanidines, a Novel Class of Nitric Oxide SynthaseInhibitors with Selectivity Towards the Inducible Isoform”, Br. J.Pharm., 117:619-32 (1996); and Szab{acute over (0)} et al.,“Mercaptoethylguanidine and Guanidine Inhibitors of Nitric OxideSynthase React with Peroxynitrite and Protect AgainstPeroxynitrite-induced Oxidative Damage”, J. Biol. Chem., 272:9030-36(1997).

[0014] Evidence also exists that PARP inhibitors are useful for treatingarthritis. Szabó et al., “Protective Effects of an Inhibitor ofPoly(ADP-Ribose)Synthetase in Collagen-Induced Arthritis,” Japanese J.Pharm., 75, Supp. I:102 (1997); Szabó et al., “DNA Strand Breakage,Activation of Poly(ADP-Ribose)Synthetase, and Cellular Energy Depletionare Involved in the Cytotoxicity in Macrophages and Smooth Muscle CellsExposed to Peroxynitrite,” Proc. Nati. Acad. Sci. USA, 93:1753-58 (March1996); and Bauer et al., “Modification of Growth Related EnzymaticPathways and Apparent Loss of Tumorigenicity of a ras-transformed BovineEndothelial Cell Line by Treatment with 5-Iodo-6-anino-1,2-benzopyrone(INH2BP)”, Intl. J. Oncol., 8:239-52 (1996); and Hughes et al.,“Induction of T Helper Cell Hyporesponsiveness in an Experimental Modelof Autoimmunity by Using Nonmitogenic Anti-CD3 Monoclonal Antibody”, J.Immuno., 153:3319-25 (1994).

[0015] Further, PARP inhibitors appear to be useful for treatingdiabetes. Heller et al., “Inactivation of the Poly(ADP-Ribose)PolymeraseGene Affects Oxygen Radical and Nitric Oxide Toxicity in Islet Cells,”J. Biol. Chem., 270:19, 11176-80 (May 1995). Heller et al. used cellsfrom mice with inactivated PARP genes and found that these mutant cellsdid not show NAD⁺ depletion after exposure to DNA-damaging radicals. Themutant cells were also found to be more resistant to the toxicity of NO.

[0016] PARP inhibitors have been shown to be useful for treatingendotoxic shock or septic shock. Zingarelli et al., “Protective Effectsof Nicotinamide Against Nitric Oxide-Mediated Delayed Vascular Failurein Endotoxic Shock: Potential Involvement of PolyADP RibosylSynthetase,” Shock, 5:258-64 (1996), suggests that inhibition of the DNArepair cycle triggered by poly(ADP ribose) synthetase has protectiveeffects against vascular failure in endotoxic shock. Zingarelli et al.found that nicotinamide protects against delayed, NO-mediated vascularfailure in endotoxic shock. Zingarelli et al. also found that theactions of nicotinamide may be related to inhibition of the NO-mediatedactivation of the energy-consuming DNA repair cycle, triggered bypoly(ADP ribose) synthetase. Cuzzocrea, “Role of Peroxynitrite andActivation of Poly(ADP-Ribose) Synthetase in the Vascular FailureInduced by Zymosan-activated Plasma,” Brit. J. Pharm., 122:493-503(1997).

[0017] PARP inhibitors have been used to treat cancer. Suto et al.,“Dihydroisoquinolinones: The Design and Synthesis of a New Series ofPotent Inhibitors of Poly(ADP-Ribose) Polymerase”, Anticancer Drug Des.,7:107-17 (1991). In addition, Suto et al., U.S. Pat. No. 5,177,075,discusses several isoquinolines used for enhancing the lethal effects ofionizing radiation or chemotherapeutic agents on tumor cells. Weltin etal., “Effect of 6(5 H)-Phenanthridinone, an Inhibitor ofPoly(ADP-ribose) Polymerase, on Cultured Tumor Cells”, Oncol. Res., 6:9,399-403 (1994), discusses the inhibition of PARP activity, reducedproliferation of tumor cells, and a marked synergistic effect when tumorcells are co-treated with an alkylating drug.

[0018] Still another use for PARP inhibitors is the treatment ofperipheral nerve injuries, and the resultant pathological pain syndromeknown as neuropathic pain, such as that induced by chronic constrictioninjury (CCI) of the common sciatic nerve and in which transsynapticalteration of spinal cord dorsal horn characterized by hyperchromatosisof cytoplasm and nucleoplasm (so-called “dark” neurons) occurs. Mao etal., Pain, 72:355-366 (1997).

[0019] PARP inhibitors have also been used to extend the lifespan andproliferative capacity of cells including treatment of diseases such asskin aging, Alzheimer's disease, atherosclerosis, osteoarthritis,osteoporosis, muscular dystrophy, degenerative diseases of skeletalmuscle involving replicative senescence, age-related musculardegeneration, immune senescence, AIDS, and other immune senescencediseases; and to alter gene expression of senescent cells. WO 98/27975.

[0020] Large numbers of known PARP inhibitors have been described inBanasik et al., “Specific Inhibitors of Poly(ADP-Ribose) Synthetase andMono(ADP-Ribosyl)-Transferase”, J. Biol. Chem., 267:3, 1569-75 (1992),and in Banasik et al., “Inhibitors and Activators of ADP-RibosylationReactions”, Molec. Cell. Biochem., 138:185-97 (1994). However, effectiveuse of these PARP inhibitors, in the ways discussed above, has beenlimited by the concurrent production of unwanted side-effects (Milam etal., “Inhibitors of Poly(Adenosine Dipbosphate-Ribose) Synthesis: Effecton Other Metabolic Processes”, Science, 223:589-91 (1984)).

[0021] There continues to be a need for effective and potent PARPinhibitors which produce minimal side-effects. The present inventionprovides compounds, compositions for, and methods of, inhibiting PARPactivity for treating and/or preventing cellular, tissue and/or organdamage resulting from cell damage or death due to, for example, necrosisor apoptosis. The compounds and compositions of the present inventionare specifically useful in ameliorating, treating and/or preventingneural tissue or cell damage, including that following focal ischemiaand reperfusion injury. Generally, inhibition of PARP activity sparesthe cell from energy loss, preventing irreversible depolarization of theneurons and, thus, provides neuroprotection. While not wishing to bebound by any mechanistic theory, the inhibition of PARP activity by useof the compounds, compositions and methods of the present invention isbelieved to protect cells, tissue and organs by protection against theill-effects of reactive free radicals and nitric oxide. The presentinvention therefore also provides methods of treating and/or preventingcells, tissue and/or organs from reactive free radical and/or nitricoxide induced damage or injury.

SUMMARY OF THE INVENTION

[0022] The present invention provides 6(5 H)phenan- or (5H)phenanthridin-6-one compounds which inhibit poly(ADP-ribose)polymerase (“PARP”), compositions containing these compounds and methodsfor making and using these PARP inhibitors to treat, prevent and/orameliorate the effects of the conditions described herein.

[0023] The compounds of the present invention are broadly described bythe following Formula I:

[0024] or a pharmaceutically acceptable salt, hydrate, prodrug, ormixtures thereof, wherein:

[0025] R₁ hydrogen or halogen;

[0026] R₂ is hydrogen, hydroxyl, amino, nitroso, methyl, amino methyl orcarboxylic acid, preferably R₁ and R₂ are not both hydrogen;

[0027] one of R₃ and R₄ is -QP and the other of R₃ and R₄ is one ofhydrogen, methyl, trifluoromethyl, nitro, amino, halogen and1-piperazine,

[0028] wherein Q is one of

[0029] P is Z-(N(R₅R₆)), such as Z-N(R₅R₆), Z or a 5 or 6 memberedsubstituted or unsubstituted aromatic or non-aromatic ring whichcontains 0, 1, 2 or 3 heteroatoms selected from the group consisting ofO, N, S and a combination of two or three of O, N and S;. wherein A iscarbon or S=O,

[0030] X is O, S , N or an N-substituted amino acid; provided that,

[0031] when X is O or S, then Y is absent,

[0032] when X is N, then Y is hydrogen, C₁-C₆ straight or branched chainalkyl, optionally substituted alkoxy or alkyl amino, or Y and Z aretaken together to form a 5, 6 or 7 membered substituted or unsubstitutedheterocyclic aromatic or non-aromatic ring which contains 1, 2 or 3heteroatoms selected from O, N, S and mixtures combination;

[0033] Z is hydrogen, a direct bond, a carbonyl, an optionallysubstituted C₁-C₅ straight or branched chain alkyl, cycloalkyl, carboxy,optionally substituted C₁-C₆ ether, aryl or heteroaryl, alkylalkenyl,alkynyl, alkylhalo, straight or branched chain or CH₂COOH, provided thatwhen P is Z, Z is not hydrogen and

[0034] R₅ and R₆ are independently hydrogen, lower alkyl, lower alkenyl,lower alkanol, heterocycle, heteroaryl, alkoxy, aryloxy, aLkylamino,arylamino, CH₂COOH, or R₅ and R₆ taken together form a 5, 6 or 7membered substituted or unsubstituted heterocyclic or cycloalkyl ringcontaining 1, 2 or 3 heteroatoms selected from O, N, S and combinationsthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035]FIG. 1 shows the distribution of the cross-sectional infarct areaat representative levels along the rostrocaudal axis, as measured fromthe interaural line in non-treated animals and in animals treated with10 mg/kg of 3,4-dihydro-5-[4- (1-piperidinyl)-botoxyl] -1 (2H)-isoquinolinone.

[0036]FIG. 2 shows the effect of intraperitoneal administration of3,4-dihydro-5-[4-(1-piperidinyl)-butoxy]-1(2 H)-isoquinolinone on theinfarct volume.

DETAILED DESCRIPTION

[0037] The present invention pertains to compounds, pharmaceuticalcompositions containing the same, methods of using the same, and processof making the same, wherein such compounds are useful as inhibitors ofpoly(ADP-ribose) polymerase (PARP). As such, they treat or preventneural tissue damage resulting from cell damage or death due to necrosisor apoptosis, cerebral ischemia and reperfusion injury orneurodegenerative diseases in an animal; they extend the lifespan andproliferative capacity of cells and thus can be used to treat or preventdiseases associated therewith; they alter gene expression of senescentcells; and they radiosensitize hypoxic tumor cells. Preferably, thecompounds of the invention treat or prevent tissue damage resulting fromcell damage or death due to necrosis or apoptosis, and/or effectneuronal activity, either mediated or not mediated by NMDA toxicity.These compounds are thought to interfere with more than the glutamateneurotoxicity and NO-mediated biological pathways. Further, thecompounds of the invention can treat or prevent other tissue damagerelated to PARP activation. The present invention provides compoundswhich inhibit poly(ADP-ribose) polymerase (“PARP”), compositionscontaining these compounds and methods for using these PARP inhibitorsto treat, prevent and/or ameliorate the effects of the conditionsdescribed herein.

[0038] In one embodiment, the present invention provides compounds ofFormula I:

[0039] or a pharmaceutically acceptable salt, hydrate, prodrug, ormixtures thereof, wherein:

[0040] R₁ hydrogen or halogen;

[0041] R₂ is hydrogen, hydroxyl, amino, nitroso, methyl, amino methyl orcarboxylic acid, preferably R₁ and R₂ are not both hydrogen;

[0042] one of R₃ and R₄ is —QP and the other of R₃ and R₄ is one ofhydrogen, methyl, trifluoromethyl, nitro, amino, halogen and1-piperazine,

[0043] wherein Q is one of

[0044] P is Z-N(R₅R₆), Z or a 5 or 6 membered substituted orunsubstituted aromatic or non-aromatic ring which contains 0, 1, 2 or 3heteroatoms selected from the group consisting of O, N, S and acombination of two or three of O, N and S;. wherein A is carbon or S=O,

[0045] X is 0, S , N or an N-substituted amino acid; provided that,

[0046] when X is O or S, then Y is absent,

[0047] when X is N, then Y is hydrogen, C₁-C₆ straight or branched chainalkyl, alkoxy or alkyl amino, or Y and Z are taken together to form a 5,6 or 7 membered substituted or unsubstituted heterocyclic aromatic ornon-aromatic ring which contains 1, 2 or 3 heteroatoms selected from O,N, S and mixtures combination;

[0048] Z is hydrogen, a direct bond, a carbonyl or an optionallysubstituted C₁-C₅ straight or branched chain alkyl, alkenyl, alkynyl,alkylhalo, or CH₂COOH, provided that when P is Z, Z is not hydrogen and

[0049] R₅ and R₆ are independently hydrogen, lower alkyl, lower alkenyl,lower alkanol, heterocycle, heteroaryl, alkoxy, aryloxy, alkylamino,arylamino, CH₂COOH, or R₅ and R₆ taken together form a 5, 6 or 7membered substituted or unsubstituted aryl, heteroaryl, heterocyclic orcycloalkyl ring containing 1, 2 or 3 heteroatoms selected from O, N, Sand combinations thereof.

[0050] In yet a further embodiment, the present invention provides thefollowing compounds of Formulas (II), (III), (IV) and (V):

[0051] wherein R₁-R₆, A and Z are as defined above.

[0052] In one embodiment, the compounds of the invention are describedby the compound of Formula I, wherein R₂ is hydrogen, Q is

[0053] and X is N. Preferred compounds of this embodiment are furtherdefined by A as carbon and P as Z-N(R₅R₆), more preferably where R₅ andR₆ form a 5 or 6 membered substituted or unsubstituted heterocycle orheteroaryl. Alternative compounds of the preferred compounds of thisembodiment are further defined by R₅ and R₆ being independently selectedfrom hydrogen, lower alkyl, alkoxy, alkylamino and CH₂COOH. Preferablyone of R₃ or R₄ is hydrogen.

[0054] In another embodiment, the compounds of the invention aredescribed by the compound of Formula I, wherein R₂ is hydrogen, Q is

[0055] and X is N and A as S=O. Preferred embodiments include compoundswherein and P is a 5 or 6 membered substituted or unsubstituted aromaticor non-aromatic ring which contains 0, 1, 2 or 3 heteroatoms selectedfrom the group consisting of O, N, S and a combination of two or threeof 0, N and S. Z in these compounds is preferably and aromatic ringcontaining 0 heteroatoms. Preferably one of R₃ or R₄ is hydrogen.

[0056] In a further embodiment, the compounds of the invention aredescribed by the compound of Formula I, wherein R₂ is hydrogen, Q is

[0057] and X is N. Preferred embodiments include compounds wherein and Zand Y are taken together to form a 5, 6 or 7 membered substituted orunsubstituted heterocyclic aromatic or non-aromatic ring, preferably anon-aromatic ring. Alternatively preferred compounds of this embodimentinclude compounds wherein P is Z-N(R₅R₆). Preferably one of R₃ or R₄ ishydrogen.

[0058] Compositions containing these preferred and alternate embodimentsand methods of making and using the same, as described herein, are alsopreferred Preferably, the compounds of the invention exhibit an IC₅₀ forinhibiting PARP in vitro, as measured by the methods described herein,of about 20 μM or less, preferably less than about 10 μM, morepreferably less than about 1 μM, or less than 0.1 μM, most preferablyless than about 0.01 μM.

[0059] Preferred embodiments of the present invention include thefollowing compounds (where compound numbers are shown next to eachcompound), and neutral forms thereof, where appropriate:

[0060] Broadly, the compounds and compositions of the present inventioncan be used to treat or prevent cell damage or death due to necrosis orapoptosis, cerebral ischemia and reperfusion injury or neurodegenerativediseases in an animal, such as a human. The compounds and compositionsof the present invention can be used to extend the lifespan andproliferative capacity of cells and thus can be used to treat or preventdiseases associated therewith; they alter gene expression of senescentcells; and they radiosensitize hypoxic tumor cells. Preferably, thecompounds and compositions of the invention can be used to treat orprevent tissue damage resulting from cell damage or death due tonecrosis or apoptosis, and/or effect neuronal activity, either mediatedor not mediated by NMDA toxicity. The compounds of the present inventionare not limited to being useful in treating glutamate mediatedneurotoxicity and/or NO-mediated biological pathways. Further, thecompounds of the invention can be used to treat or prevent other tissuedamage related to PARP activation, as described herein.

[0061] The present invention provides compounds which inhibit the invitro and/or in vivo polymerase activity of poly(ADP-ribose) polymerase(PARP), and compositions containing the disclosed compounds.

[0062] The present invention provides methods to inhibit, limit and/orcontrol the in vitro and/or in vivo polymerase activity ofpoly(ADP-ribose) polymerase (PARP) in any of solutions, cells, tissues,organs or organ systems. In one embodiment, the present inventionprovides methods of limiting or inhibiting PARP activity in a mammal,such as a human, either locally or systemically.

[0063] The present invention provides methods to treat and/or preventdiseases, syndromes and/or conditions exacerbated by or involving theincreased generation of PARP. These methods involve application oradministration of the compounds of the present invention to cells,tissues, organs or organ systems of a person in need of such treatmentor prevention.

[0064] In one embodiment, the present invention provides methods totreat and/or prevent cardiovascular tissue damage resulting from cardiacischemia or reperfusion injury. Reperfusion injury, for instance, occursat the termination of cardiac bypass procedures or during cardiac arrestwhen the heart, once prevented from receiving blood, begins to reperfuseand these methods involve administration of the compounds andcompositions of the present invention preferably prior to, orimmediately subsequent to reperfusion, such that reperfusion injury isprevented, treated or reduced. The present invention also providesmethods of preventing and/or treating vascular stroke, cardiovasculardisorders

[0065] In another embodiment, the present invention provides in vitro orin vivo methods to extend or increase the lifespan and/or proliferationcapacity of cells and thus also methods to treat and/or prevent diseasesassociated therewith and induced or exacerbated by cellular senescenceincluding skin aging, atherosclerosis, osteoarthritis, osteoporosis,muscular dystrophy, degenerative diseases of skeletal muscle involvingreplicative senescence, age-related muscular degeneration, immunesenescence, AIDS and other immune senescence diseases, and otherdiseases associated with cellular senescence and aging, as well as toalter the gene expression of senescent cells.

[0066] In a further embodiment, the present invention provides methodsof treating or preventing or ameliorating the effect of cancer and/or toradiosensitize hypoxic tumor cells to render the tumor cells moresusceptible to radiation therapy and thereby to prevent the tumor cellsfrom recovering from potentially lethal damage of DNA after radiationtherapy. A method of this embodiment is directed to specifically andpreferentially radiosensitizing tumor cells rendering the tumor cellsmore susceptible to radiation therapy than non-tumor cells.

[0067] In yet another embodiment the present invention provides methodsof preventing and/or treating vascular stroke, cardiovascular disorders;to treat other conditions and/or disorders such as age-related musculardegeneration, AIDS and other immune senescence diseases, inflammation,arthritis, gout, atherosclerosis, cachexia, cancer, degenerativediseases of skeletal muscle involving replicative senescence,hyperglycemia, diabetes, head trauma, spinal chord injury, immunesenescence, gout, inflammatory bowel disorders (such as colitis andCrohn's disease), acute pancreatitis, mucositis, hemorrhagic shock,splanchnic artery occlusion shock, multiple organ failure (such asinvolving any of the kidney, liver, renal, pulmonary, retinal,pancreatic and/or skeletal muscles systems), acute autoimmunethyroiditis, muscular dystrophy, osteoartritis, osteoporosis, chronicand/or acute pain (such as neuropathic pain), renal failure, retinalischemia, septic shock (such as endotoxic shock), local and/or remoteendothelial cell dysfunction (such are recognized by endo-dependentrelaxant responses and up-regulation of adhesion molecules),inflammation and skin aging.

[0068] The compounds of the present invention may be administered, forexample, parenterally, to a person diagnosed with acute retinalischeniia or acute vascular stroke, either by intermittent or continuousintravenous administration, by either a single dose or a series ofdivided doses. Compounds of the invention may be used in combination orsequentially. The compound of the invention can be administered byintermittent or continuous administration via implantation of abiocompatible, biodegradable polymeric matrix delivery system containinga compound of formula I, II, III, IV or V, or via a subdural pumpinserted to administer the compound directly to the infarct area of thebrain.

[0069] In a further embodiment, the present invention provides methodsto extend the lifespan and proliferative capacity of cells, such as, forexample, in using the compounds of the invention as general mediators inthe generation of oxidants, proinflammatory mediators and/or cytokines,and/or general mediators of leukocyte infiltration, calcium ionoverload, phospholipid peroxidation, impaired nitric oxide metabolismand/or reduced ATP production

[0070] For example, the compounds of the invention can treat or preventcardiovascular tissue damage resulting from cardiac ischemia orreperfilsion injury. Reperfusion injury, for instance, occurs at thetermination of cardiac bypass procedures or during cardiac arrest whenthe heart, once prevented from receiving blood, begins to reperfuse.

[0071] The compounds of the present invention can also be used to extendor increase the lifespan or proliferation of cells and thus to treat orprevent diseases associated therewith and induced or exacerbated bycellular senescence including skin aging, atherosclerosis,osteoarthritis, osteoporosis, muscular dystrophy, degenerative diseasesof skeletal muscle involving replicative senescence, age-relatedmuscular degeneration, immune senescence, AIDS and other immunesenescence diseases, and other diseases associated with cellularsenescence and aging, as well as to alter the gene expression ofsenescent cells. These compounds can also be used to treat cancer and toradiosensitize hypoxic tumor cells to render the tumor cells moresusceptible to radiation therapy and to prevent the tumor cells fromrecovering from potentially lethal damage of DNA after radiationtherapy, presumably by their ability to prevent DNA repair. Thecompounds of the present invention can be used to prevent or treatvascular stroke; to treat or prevent cardiovascular disorders; to treatother conditions and/or disorders such as age-related musculardegeneration, AIDS and other immune senescence diseases, inflammation,arthritis, gout, atherosclerosis, cachexia, cancer, degenerativediseases of skeletal muscle involving replicative senescence,hyperglycemia, diabetes, head trauma, immune senescence, gout,inflammatory bowel disorders (such as colitis and Crohn's disease),muscular dystrophy, osteoarthritis, osteoporosis, chronic and/or acutepain (such as neuropathic pain), renal failure, retinal ischemia, septicshock (such as endotoxic shock), and skin aging.

[0072] Preferably, the compounds of the invention act as PARP inhibitorsto treat or prevent tissue damage resulting from cell death or damagedue to necrosis or apoptosis; to treat or prevent neural tissue damageresulting from cerebral ischemia and reperfiusion injury orneurodegenerative diseases in an animal; to extend and increase thelifespan and proliferative capacity of cells; to alter gene expressionof senescent cells; and to radiosensitize tumor cells.

[0073] Another especially preferred embodiment of the invention is apharmaceutical composition which comprises (i) a therapeuticallyeffective amount of the compound of fonnula I, II, III, IV or V; and(ii) a pharnaceutically acceptable carrier.

[0074] As used herein, “alkyl” means a branched or unbranched saturatedhydrocarbon chain comprising a designated number of carbon atoms. Forexample, C₁-C₆ straight or branched alkyl hydrocarbon chain contains 1to 6 carbon atoms, and includes but is not limited to substituents suchas methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, tert-butyl,n-pentyl, n-hexyl, and the like, unless otherwise indicated.

[0075] Optional substitutions of alkyl and ether chains includemercapto, carboxy, hydroxy, or phenyl, benzyl, or phenylethyl, which maythemselves be substituted by hydroxy, halo, methoxy, C₁-C₆ alkyl, amineand carboxy.

[0076] “Alkenyl” means a branched or unbranched unsaturated hydrocarbonchain comprising a designated number of carbon atoms. For example, C₂-C₆straight or branched alkenyl hydrocarbon chain contains 2 to 6 carbonatoms having at least one double bond, and includes but is not limitedto substituents such as ethenyl, propenyl, isopropenyl, butenyl,iso-butenyl, tert-butenyl, n-pentenyl, n-hexenyl, and the like, unlessotherwise indicated.

[0077] “Alkoxy”, means the group —OR wherein R is alkyl as hereindefined. Preferably, R is a branched or unbranched saturated hydrocarbonchain containing 1 to 6 carbon atoms.

[0078] “Cyclo”, used herein as a prefix, refers to a structurecharacterized by a closed ring.

[0079] “Halo” means at least one fluoro, chloro, bromo, or iodo moiety,unless otherwise indicated.

[0080] “Amino” compounds include amine (NH₂) as well as substitutedamino groups comprising alkyls of one through six carbons.

[0081] “Ar”, “aryl” or “heteroaryl” means a moiety which is substitutedor unsubstituted, especially a cyclic or fused cyclic ring and includesa mono-, bi-, or tricyclic, carbo- or heterocyclic ring, such as a 5, 6,7 or 8 membered ring, wherein the ring is either unsubstituted orsubstituted in, for example, one to five position(s) with halo,haloalkyl, hydroxyl, nitro, trifluoromethyl, C₁-C₆ straight or branchedchain alkyl, C₂-C₆ straight or branched chain alkenyl, C₁-C₆ alkoxy,-C(O)-O(C₁-C₆ allyl), carboxy, C₂-C₆ alkenyloxy, phenoxy, benzyloxy,amino, thiocarbonyl, ester, thioester, cyano, imino, alkylamino,aminoalkyl, sulfhydryl, thioalkyl, and sulfonyl; wherein the individualring sizes are preferably 5-8 members; wherein the heterocyclic ringcontains 1-4 heteroatom(s) selected from the group consisting of O, N,or S or their mixture; wherein aromatic or tertiary aLkyl amines areoptionally oxidized to a corresponding N-oxide. Heteroaryls may beattached to other rings or substituted through the heteroatom and/orcarbon atom of the ring. Particularly preferred aryl or heteroarylmoieties include but are not limited to phenyl, benzyl, naphthyl,piperidino, pyrrolyl, pyrrolidinyl, pyridinyl, pyrimidinyl, purinyl,quinolinyl, isoquinolinyl, furyl, thiophenyl, imidazolyl, oxazolyl,thiazolyl, pyrazolyl, and thienyl.

[0082] “Phenyl” includes all possible isomeric phenyl radicals,optionally monosubstituted or multi-substituted with substituentsselected from the group consisting of amino, trifluoromethyl, C₁-C₆straight or branched chain alkyl, C₂-C₆ straight or branched chainaLkenyl, carbonyl, thiocarbonyl, ester, thioester, alkoxy, aLkenoxy,cyano, nitro, imino, alkylamino, aminoalkyl, sulfhydryl, thioalkyl,sulfonyl, hydroxy, halo, haloalkyl, NR₂ wherein R₂ is selected from thegroup consisting of hydrogen, (C₁-C₆)-straight or branched chain aLkyl,(C₃-C₆) straight or branched chain alkenyl or alkynyl and 2, 3 or 4fused phenyl rings.

[0083] Cycloalkyl optionally containing at least one heteroatom, to forma heterocyclic ring, includes saturated C₃-C₈ rings, preferably C₅ or C₆rings, wherein at 1, 2, 3 or 4 heteroatoms selected from O, N or S maybe optionally substituted for a carbon atom of the ring. Cycloalkylsoptionally containing at least one heteroatom, as described above, maybe substituted by or fused to at least one 5 or 6 membered aryl orheteroaryl and/or substituted by at least one of amino, C₁ -C₅ straightor branched chain alkyl, Cl -C₆ alkanol, C₁ -C₆ straight or branchedchain alkylamino, C₁-C₆ alkoxy, or C₁-C₆ alkenyl, or benzyl, or phenylor phenylethyl wherein the ring may be substituted as described abovefor substitutions of “Phenyl”.

[0084] Preferred cycloalkyls containing at least one heteroatom include

[0085] pyrrolidinyl, indolyl, 2,3-dihydro-1 H isoindolyl,imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, morpholino andthiomorpholino.

[0086] The compounds of the present invention possess one or moreasymmetric center(s) and thus can be produced as mixtures (racemic andnon-racemic) of stereoisomers, or as individual enantiomers ordiastereomers. The individual stereoisomers may be obtained by using anoptically active starting material, by resolving a racemic ornon-racemic mixture of an intermediate at some appropriate stage of thesynthesis, of by resolution of the compound of any of formulas I, II,III, IV and V. It is understood that the individual stereoisomers aswell as mixtures (racemic and non-racemic) of stereoisomers areencompassed by the scope of the present invention.

[0087] The compounds of the invention are useful in a free base form, inthe form of pharmaceutically acceptable salts, pharmaceuticallyacceptable hydrates, pharmaceutically acceptable esters,pharmaceutically acceptable solvates, pharmaceutically acceptableprodrugs, pharmaceutically acceptable metabolites, and in the form ofpharmaceutically acceptable stereoisomers. These forms are all withinthe scope of the invention. In practice, the use of these forms amountsto use of the neutral compound.

[0088] “Pharmaceutically acceptable salt”, “hydrate”, “ester” or“solvate” refers to a salt, hydrate, ester, or solvate of the inventivecompounds which possesses the desired pharmacological activity and whichis neither biologically nor otherwise undesirable. Organic acids can beused to produce salts, hydrates, esters, or solvates such as acetate,adipate, alginate, aspartate, benzoate, benzenesulfonate,p-toluenesulfonate, bisulfate, sulfamate, sulfate, naphthylate,butyrate, citrate, camphorate, camphorsulfonate,cyclopentane-propionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, glucoheptanoate, glycerophosphate, hemisulfate heptanoate,hexanoate, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate,2-naphthalenesulfonate, nicotinate, oxalate, tosylate and undecanoate.Inorganic acids can be used to produce salts, hydrates, esters, orsolvates such as hydrochloride, hydrobromide, hydroiodide, andthiocyanate.

[0089] Examples of suitable base salts, hydrates, esters, or solvatesinclude hydroxides, carbonates, and bicarbonates of ammonia, alkalimetal salts such as sodium, lithium and potassium salts, alkaline earthmetal salts such as calcium and magnesium salts, aluminum salts, andzinc salts.

[0090] Salts, hydrates, esters, or solvates may also be formed withorganic bases. Organic bases suitable for the formation ofpharmaceutically acceptable base addition salts, hydrates, esters, orsolvates of the compounds of the present invention include those thatare non-toxic and strong enough to form such salts, hydrates, esters, orsolvates. For purposes of illustration, the class of such organic basesmay include mono-, di-, and trialkylamines, such as methylamine,dimethylamine, triethylamnine and dicyclohexylamine; mono-, di- ortrihydroxyalkylarnines, such as mono-, di-, and triethanolarnine; aminoacids, such as arginine and lysine; guanidine; N-methyl-glucosamine;N-methyl-glucarnine; L-glutamine; N-methyl-piperazine; morpholine;ethylenediamine; N-benzyl-phenethylamine;(trihydroxy-methyl)aminoethane; and the like. See, for example,“Pharmaceutical Salts,” J. Pharm. Sci., 66:1, 1-19 (1977). Accordingly,basic nitrogen-containing groups can be quaternized with agentsincluding: lower alkyl halides such as methyl, ethyl, propyl, and butylchlorides, bromides and iodides; dialkyl sulfates such as dimethyl,diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl,lauryl, myristyl and stearyl chlorides, bromides and iodides; andaralkyl halides such as benzyl and phenethyl bromides.

[0091] The acid addition salts, hydrates, esters, or solvates of thebasic compounds may be prepared either by dissolving the free base of aPARP inhibitor of the present invention in an aqueous or an aqueousalcohol solution or other suitable solvent containing the appropriateacid or base, and isolating the salt by evaporating the solution.Alternatively, the free base of the PARP inhibitor of the presentinvention can be reacted with an acid, as well as reacting the PARPinhibitor having an acid group thereon with a base, such that thereactions are in an organic solvent, in which case the salt separatesdirectly or can be obtained by concentrating the solution.

[0092] “Pharmaceutically acceptable prodrug” refers to a derivative ofthe inventive compounds which undergoes biotransformation prior toexhibiting its pharmacological effect(s). The prodrug is formulated withthe objective(s) of improved chemical stability, improved patientacceptance and compliance, improved bioavailability, prolonged durationof action, improved organ selectivity, improved formulation (e.g.,increased hydrosolubility), and/or decreased side effects (e.g.,toxicity). The prodrug can be readily prepared from the inventivecompounds using methods known in the art, such as those described byBurger's Medicinal Chemistry and Drug Chemistry, Fifth Ed., Vol, 1, pp.172-178, 949-982 (1995). For example, the inventive compounds can betransformed into prodrugs by converting one or more of the hydroxy orcarboxy groups into esters.

[0093] “Pharmaceutically acceptable metabolite” refers to drugs thathave undergone a metabolic transformation. After entry into the body,most drugs are substrates for chemical reactions that may change theirphysical properties and biologic effects. These metabolic conversions,which usually affect the polarity of the compound, alter the way inwhich drugs are distributed in and excreted from the body. However, insome cases, metabolism of a drug is required for therapeutic effect. Forexample, anticancer drugs of the antimetabolite class must be convertedto their active forms after they have been transported into a cancercell. Since most drugs undergo metabolic transformation of some kind,the biochemical reactions that play a role in drug metabolism may benumerous and diverse. The main site of drug metabolism is the liver,although other tissues may also participate.

[0094] The term “neurodegenerative diseases” includes Alzheimer'sdisease, Parkinson's disease and Huntington's disease.

[0095] The term “nervous insult” refers to any damage to nervous tissueand any disability or death resulting therefrom. The cause of nervousinsult may be metabolic, toxic, neurotoxic, iatrogenic, thermal orchemical, and includes without limitation, ischemia, hypoxia,cerebrovascular accident, trauma, surgery, pressure, mass effect,hemmorrhage, radiation, vasospasm, neurodegenerative disease, infection,Parkinson's disease, amyotrophic lateral sclerosis (ALS),myelination/demyelination process, epilepsy, cognitive disorder,glutamate abnormality and secondary effects thereof.

[0096] The term “neuroprotective” refers to the effect of reducing,arresting or ameliorating nervous insult, and protecting, resuscitating,or reviving nervous tissue that has suffered nervous insult.

[0097] The term “preventing neurodegeneration” includes the ability toprevent neurodegeneration in patients diagnosed as having aneurodegenerative disease or who are at risk of developing aneurodegenerative disease. The term also encompasses preventing furtherneurodegeneration in patients who are already suffering from or havesymptoms of a neurodegenerative disease.

[0098] The term “treating” refers to:

[0099] (i) preventing a disease, disorder or condition from occurring inan animal that may be predisposed to the disease, disorder and/orcondition, but has not yet been diagnosed as having it;

[0100] (ii) inhibiting the disease, disorder or condition, i.e.,arresting its development; and

[0101] (iii) relieving the disease, disorder or condition, i.e., causingregression of the disease, disorder and/or condition.

[0102] The term “neural tissue damage resulting from ischemia andreperfiision injury and neurodegenerative diseases” includesneurotoxicity, such as seen in vascular stroke and global and focalischemia.

[0103] A feature characteristic of many of these transformations is thatthe metabolic products are more polar than the parent drugs, although apolar drug does sometimes yield a less polar product. Substances withhigh lipid/water partition coefficients, which pass easily acrossmembranes, also diffuse back readily from tubular urine through therenal tubular cells into the plasma. Thus, such substances tend to havea low renal clearance and a long persistence in the body. If a drug ismetabolized to a more polar compound, one with a lower partitioncoefficient, its tubular reabsorption will be greatly reduced. Moreover,the specific secretory mechanisms for anions and cations in the proximalrenal tubules and in the parenchymal liver cells operate upon highlypolar substances.

[0104] As a specific example, phenacetin (acetophenetidin) andacetanilide are both mild analgesic and antipyretic agents, but are eachtransformed within the body to a more polar and more effectivemetabolite, p-hydroxyacetanilid (acetaminophen), which is widely usedtoday. When a dose of acetanilid is given to a person, the successivemetabolites peak and decay in the plasma sequentially. During the firsthour, acetanilid is the principal plasma component. In the second hour,as the acetanilid level falls, the metabolite acetaminophenconcentration reaches a peak. Finally, after a few hours, the principalplasma component is a further metabolite that is inert and can beexcreted from the body. Thus, the plasma concentrations of one or moremetabolites, as well as the drug itself, can be pharmacologicallyimportant.

[0105] The reactions involved in drug metabolism are often classifiedinto two groups, as shown in the Table II. Phase I (orfunctionalization) reactions generally consist of (1) oxidative andreductive reactions that alter and create new functional groups and (2)hydrolytic reactions that cleave esters and amides to release maskedfunctional groups. These changes are usually in the direction ofincreased polarity.

[0106] Phase II reactions are conjugation reactions in which the drug,or often a metabolite of the drug, is coupled to an endogenoussubstrate, such as glucuronic acid, acetic acid, or sulfuric acid. TABLEII Phase I Reactions (functionalization reactions): (1) Oxidation viathe hepatic microsomal P450 system: Aliphatic oxidation Aromatichydroxylation N-Dealkylation O-Dealkylation S-Dealkylation EpoxidationOxidative deamination Sulfoxide formation Desulfuration N-Oxidation andN-hydroxylation Dehalogenation (2) Oxidation via nonmicrosomalmechanisms: Alcohol and aldehyde oxidation Purine oxidation Oxidativedeamination (monoamine oxidase and diamine oxidase) (3) Reduction: Azoand nitro reduction (4) Hydrolysis: Ester and amide hydrolysis Peptidebond hydrolysis Epoxide hydration Phase II Reactions (conjugationreactions): (1) Glucuronidation (2) Acetylation (3) Mercapturic acidformation (4) Sulfate conjugation (5) N-, O-, and S-methylation (6)Trans-sulfuration

[0107] The compounds of the present invention exhibit pharmacologicalactivity and are, therefore, usefil as pharmaceuticals. In particular,the compounds exhibit central nervous and cardiac vesicular systemactivity. It is understood that tautomeric forms, when possible, areincluded in the invention.

[0108] Many of the PARP inhibitors are known and, thus, can besynthesized by known methods from starting materials that are known, maybe available commercially, or may be prepared by methods used to preparecorresponding compounds in the literature. See, for example, Suto etal., “Dihydroiso-quinolinones: The Design and Synthesis of a New Seriesof Potent Inhibitors of Poly(ADP-ribose) Polymerase”, Anticancer DrugDes., 6:107-17 (1991), which discloses processes for synthesizing anumber of different PARP inhibitors.

[0109] Typically, the PARP inhibitors used in the composition of theinvention will have an IC₅₀ for inhibiting poly(ADP-ribose) polymerasein vitro of about 20 IM or less, preferably less than about 10 μM, morepreferably less than about 1 μM, or preferably less than about 0.1 μM,most preferably less than about 0.01 μM.

[0110] The PARP inhibitor 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone, for example, has been reported to inhibit PARP withan IC₅₀ of 40 nM by Suto et al., cited above.

[0111] The 6(5 H)-phenanthridinone sulfonamides and carbamides, such asExample compounds 1-14 below, of this invention are represented bypreviously defined formula I-V. As an example, these 6(5H)-phenanthridinone derivatives can be prepared in a conventional manneras illustrated below by Schemes 1 - 3. The process sequence set forthherein does not present an exact sequence of reactions by which thecompound must be made; that is, the sequence of reactions can berearranged in several ways, by means known to those of ordinary skill inthe art, to reach the target molecule.

[0112] Scheme 1 below illustrates schematically the preparation ofcompounds Example 1 through Example 4. Compound 3 (of this scheme),2-amino-6(5 H)-phenanthridinone, is used as a precursor to prepare thedesired N-substituted 6(5 H)-phenanthridinone sulfonamides andcarbamides. Preparation of this amino derivative 3 is known in thechemistry literature and accessible by processes known to one ofordinary skill in the art. For example, nitration of commerciallyavailable compound of 6(5 H)-phenanthridinone 1 provides compound2-nitro-6(5 H)-phenanthridinone 2 which can be reduced by hydrogenationusing palladium as catalyst or by hydrazine to afford 2-amino-6(5H)-phenanthridinone 3. Target compounds-sulfonamides and carbamides canbe formed by reaction the amino group of compound 3 with either sulfonylchloride or carboxylic acid halide derivatives as described in Generalprocedure A. Formation of sulfonamides and carbamides with aminoderivatives can be carried out by a variety of conditions known to thoseof ordinary skill in the art, including reaction with first or secondaryamines using pyridine or triethyl amine as base. Typical solventsinclude chlorinated solvents, various ethers, and dipolar aproticsolvents like DMF.

[0113] Scheme 2 below illustrates schematically the preparation ofcompounds Example 10 through Example 12. Monosulfonation of commerciallyavailable 6(5 H)-phenanthridinone 1 can be achieved using chorosulfonicacid neat to give compound 4 in high yield. For example, 6(5H)-phenanthridinone 1 was placed in neat chorosulfonic acid undernitrogen at 0° C. The resulting mixture is allowed to warm to roomtemperature, continuously stirred for about 2 days and poured onto 100 gof ice. The residue is collected by filtration and washed with water,ethanol to afford the product 4 as solid without further purification,mp 317-320, ¹H-NMR (400 MHz, DMSO-d₆), 11.78 (br, 1 H), 8.50 (s, 1 H),8.41 (d, J=8.0 Hz, 1 H), 8.33 (d,J=8.0 Hz, 1 H), 7.88 (t, J=8.0 Hz, 1H), 7.72 (d, J=8.0 Hz, 1 H), 7.66 (t, J=8.0 Hz, 1 H), 7.33 (d, J=8.0 Hz,1 H). Aminmation of compound 4 using primary amines can be carried outby a variety of conditions similar to the process described above fromcompound 3 to Examples 1-9. Thus final products of compounds Examples10-12 can be prepared from 6(5 H)-phenanthridinone 1 using Generalprocedure A bellow.

[0114] Scheme 3 below illustrates schematically the preparation ofcompounds Example 13 through Example 19. The 3-substituted (5H)phenanthridin-6-one skeleton of this invention can be constructed in aconventional manner using Schmidt method with fluoren-9-one 5 asstarting material and sodium azide as an insertion agent in acidiccondition (Chida, N.: Ohtsuka. M.: Ogawa, S. Tetrahedron Lett. 1991, 32,4525). The fluoren-9-one ring may be generically substituted as setforth in the drawing. Such fluoren-9-one starting derivatives are knownin the chemistry literature and accessible by processes known to oneskilled in the art. This Schmidt method normally generates a pair ofregioisomer mixtures 6 and 7, which may be separated and purified bychromatography or crystallization. For example, a mixture of2(fluoren-9-one)carboxylic acid (0.25g) and sodium azide (0.25 g) inc-sulfric acid (10 mL) was stirred at 0° C. for 30 minutes at 25° C. for10 hours, and then poured into ice-cold water (100 mL). A yellowprecipitation appeared upon adjusting the solution to pH 7 with sodiumhydroxide. The precipitation, a regioisomer mixture of 3-[6(5H)phenanthridinone] carboxylic acid 6 and of 8-[6(5 H)phenanthridinone]carboxylic acid 7, was collected by filtration and purified on silicagel column to give a pure isomer of 3-[6(5 H)phenanthridinone]carboxylic acid 6 (0.1 g), mp 300° C. (dec.). ¹H-NMR (400 MHz, DMSO-d₆),13.19 (bs, 1 H), 11.88 (s, 1 H), 8.58 (d, 1 H, J=8 Hz), 8.52 (d, 1 H,J=8 Hz), 8.35 (d, 1 H, J=8 Hz), 8.01 (s, 1 H), 7.89 (t, 1 H, J=8 Hz),7.76 (t, 1 H, J=8 Hz), 7.72 (d, 1 H, J=8 Hz). There are several ways,such as Ullman reactions (Synthesis, 1997, p.1273-1275 “Synthesis andCharacterization of Some Nitrobenzanthrones: Suspected New Mutagens inAtmospheric Environment”, Hitomi Suzuki, Takeji Enya, YoshiharuHizamatsu) and Suzuki couplings (J. Organic. Chememistry., 1992, 57,p.379-381. “A simple asymetric sythesis of 4-arylphenylalanines vispalladium-catalyzed cross coupling reaction of arylboric acids withtyrosine triflate” W.-C. Shieh; J. A. Carlson), to construct substituted6(5 H)Phenanthridinone carboxylic acid and 6(5 H)Phenanthridinonesulfonyl chloride. Formation of substituted 6(5 H)Phenanthridinone usingbiphenyl compounds can be carried out by a variety of conditions knownto those skilled in the art. 3-[6(5 H)Phenanthridinone] carboxylic acid6 can be routinely converted to 3-[6(5 H)Phenanthridinone] carbonylchloride 7 using thionyl chloride neat at 60° C. Amidation of desiredregioisomer of 3-[6(5 H)phenanthridinone] sulfonyl chloride 6 or 3-[6(5H)Phenanthridinone]carbonyl chloride 7 with R′ substituted primary amineusing triethyl amine (TEA) as base provides (5 H)phenanthridin-6-onesulfonamide and carbamide of Example 13-19.

[0115] General procedure A:

[0116] To a suspension of the sulfonyl chloride or compound 6 (1.1 mmol)in methylene chloride (100 mL) or p-dioxane (100 mL) is added triethylamine (1.5 mmol) and amine compound or compound 3 (1.0 mmol) undernitrogen at 0° C. The reaction mixture is allowed to warm to roomtemperature, stirred continuously for 3 hour or until TLC showing nostarting material left and poured into 100 mL of water. The organiclayer is collected, washed with water, brine and concentrated in vacuo.The product is purified via crystallization or silica columnchromatography using methanol/methylene chloride as eluent to affordsolid product in 60-90% of yield.

EXAMPLE 1

[0117]

[0118] N-(5,6-Dihydro-6-oxo-2-phenanthridinyl)-4-methylbenzenesulfonamide

[0119] Prepared from 2-amino-6(5 H)-phenanthridinone 3 and 4-methylbenzenesulfonamide according to General Procedure A. P-dioxane was usedas solvent and the reaction was run at 40° C. for 30 hours. Purificationof compound by crystallization in dioxane gave a brown solid ( 93 %yield). mp 199-203° C. ¹H-NMR (400 MHz, DMSO-d₆), 10.20 (s, 1 H), 8.30(d, J=8.0 Hz, 1 H), 8.15 (d, J=8.1 Hz, 1 H), 7.95 (s, 1 H), 7.88 (t,J=7.6, 1 H), 7.65 (m, 3 H), 7.33 (d, J=8.2 Hz, 2 H), 7.19 (m, 2 H).Anal. (C₂₀ H₁₆ N₂ O₃ S), C H N.

EXAMPLE 2

[0120]

[0121] 2-Chloro-N-(5,6-dihydro-6-oxo-2-phenanthridinyl)acetamide

[0122] Prepared from 2-amino-6(5 H)-phenanthridinone 3 and chloroacetylchloride according to General Procedure A. P-dioxane was used as solventand 1.1 equivalent of sodium hydroxide as base. A white precipitationwas formed after stirring the mixture for 2 ours. Purification of theprecipitation by crystallization in dioxane gave a white solid (53%yield), mp 300-303° C. ¹H-NMR (400 MHz, DMSO-d₆), 11.71 (s, 1 H), 10.45(s, 1 H), 8.63 (s, 1 H), 8.33 (d, J=7.9 Hz, 1 H), 8.27 (d, J=8.2 Hz, 1H), 7.89 (t, J=7.6 Hz, 1 H), 7.64 (m, 2 H), 7.34 (d, J=8.8 Hz, 1 H),4.30 (s, 2 H). Anal. (C₁₅ H₁₁ Cl N₂ O₂), C H N.

EXAMPLE 3

[0123]

[0124]4-[(5,6-Dihydro-6-oxo-2-phenanthridinyl)amino]-4-oxo-butanoic acid

[0125] Prepared from 2-amino-6(5 H)-phenanthridinone 3 and succinicanhydride. To a solution of 2-amino-6(5 H)-phenanthridinone (150 mg,0.71 mmol) in dioxane (30 mL) was added a solution of succinic anhydride(87 mg, 1.1 mmol) in dioxane (5 mL) over 5 minutes. The mixture wasstirred for 16 hours and a yellowish precipitation was formed.Purification of the precipitation by crystallization in dioxane gave alight white solid (92% yield), mp 224-228° C. ¹H-NMR (400 MHz DMSO-d₆),11.7 (s, 1 H), 10.1 (s, 1 H), 8.67 (s, 1 H), 8.33 (d, J=7.8 Hz, 1 H),8.24 (d, J=8.2 Hz, 1 H), 7.87 (t, J=7.0 Hz, 1 H), 7.66 (t, J=7.44 Hz, 1H ), 7.60 (d, J=8.7 Hz, 1 H),7.30 (d, J=8.7 Hz, 1 H), 2.60 ((d, J=5.4Hz, 2 H), 2.56 (d, J=5.4 Hz, 2 H). Anal. (C₁₇ H₂₄ N₂ O₄ 0.1 H₂O), C H N.

EXAMPLE 4

[0126]

[0127]N-(5,6-Dihydro-6-oxo-2-phenanthridinyl)-2-(trimethyl-azanyl)-acetamidechloride

[0128] Prepared from the compound of Example 2 and trimethyl amine. To awater solution of trimethyl amine (70 mg) was added2-chloro-N-(5,6-dihydro-6-oxo-2-phenanthridinyl)acetanide (286 mg) indimethylsulfoxide (0.2 ML) at room temperature over 0.5 hours. Acetone(10 mL) was added to the mixture which was continuously stirred for 18hours. A white precipitation was collected by filtration. Purificationof the crude precipitation by crystallization in ethanol gave a whitesolid as a hydrogen chloride salt (lOOmg), mp 269-272° C (dec.). ¹H-NMR(400 MHz, DMSO-d₆), 11.76 (s, 1 H), 11.03 (s, 1 H), 8.68 (s, 1 H), 8.34(d, J=8 Hz, 1 H), 8.27 (d, J=8 Hz, 1 H), 7.91 (t, J=8 Hz, 1 H), 7.65 (m,2 H), 7.37 (d, J=8 Hz, 1 H), 4.41 (s, 2 H). Anal. (C₁₈ H₂₀ Cl N₃ O₂ 0.7H₂O), C H N.

EXAMPLE 5

[0129]

[0130]N-(5,6-Dihydro-6-oxo-2-phenanthridinyl)-2-(dimethylamino)-acetamide

[0131] Prepared from the compound of Example 2 and dimethylamine. To asolution of 2-chloro-N-(5,6-dihydro-6-oxo-2-phenanthridinyl)-acetamide(635 mg) in N, N-dimethylformamide (100 mL) was added potassiumphosphate (918 mg) and dimethylamine (1.3 ml, 2 molar soln.). Thesolution stirred at room temperature for 5 hours followed by evaporationof the solvent. The light brown product was washed with water andfiltered. Concentrated HCl (0.3 mL) was added to a solution of theproduct in dioxane (150 mL) to precipitate the hydrogen chloride salt(400 mg), mp>300° C. ¹H-NMR (300 MHz, DMSO-d₆), 11.63 (br s, 1 H), 10.92(br s, 1 H), 8.63 (s, 1 H), 8.35 (d, J=9.0 Hz, 1 H), 8.26 (d, J=9.0 Hz,1 H), 7.91 (t, J=9.0 Hz, 1 H), 7.69 (m, 2 H), 7.38 (d, J=9.0 Hz, 1 H),4.19 (br s, 2 H), 2.91 (br s, 6 H). Anal. (C₁₇H₁₇N₃O₂ HCl 0.9H₂O), Calcfor: C, 58.67; H, 5.73; N, 12.07; Cl, 10.19. Found: C, 58.91; H, 5.59;N, 11.80; Cl, 9.95.

EXAMPLE 6

[0132]

[0133]N-(5,6-Dihydro-6-oxo-2-phenanthridinyl)-2-(dipropylamino)-acetamide

[0134] Prepared from the compound of Example 2 and dipropylamine. To asolution of 2-chloro-N-(5,6-dihydro-6-oxo-2-phenanthridinyl)-acetamide(45 mg) in N, N-dimethylformamide (10 mL) was added potassium phosphate(68 mg) and dipropylamine (0.027 mL). The solution stirred at roomtemperature for 5 hours followed by evaporation of the solvent. Thelight brown product was washed with water and filtered (25 mg). ¹H-NMR(300 MHz, DMSO-d₆), 11.70 (br s, 1 H), 9.72 (br s, 1 H), 8.33 (d, J=8.0Hz, 2 H), 7.89 (m, 1), 7.66 (m, 2 H), 7.33 (m, 1 H), 3.21 (s, 2 H), 1.50(m, 6 H), 0.89 (m, 8 H).

EXAMPLE 7

[0135]

[0136] N-(5,6-Dihydro-6-oxo-2-phenanthridinyl)-1,3 -dihydro-2H-isoindole-2-acetamide

[0137] Prepared from the compound of Example 2 and isoindoline. To asolution of 2-chloro-N-(5,6-dihydro-6-oxo-2-phenanthridinyl)-acetamide(45 mg) in N, N-dimethylformamide (10 mL) was added potassium phosphate(68 mg) and isoindoline (0.025 mL). The solution stirred at roomtemperature for 5 hours followed by evaporation of the solvent. Thelight brown product was washed with water and filtered (35 mg). ¹H-NMR(300 MHz, DMSO-d₆), 11.67 (br s, 1 H), 9.97 (br s, 1 H), 8.69 (s, 1 H),8.31 (d, J=9.0 Hz, 2 H), 7.84 (m, 2 H), 7.65 (t, J=9.0 Hz, 1 H), 7.27(m, 5 H), 4.11 (s, 4 H), 3.60 (s, 2 H).

EXAMPLE 8

[0138]

[0139]2-(cyclohexylmethylamino)-N-(5,6-dihydro-6-oxo-2-phenanthridinyl)-acetamide

[0140] Prepared from the compound of Example 2 andN-methylcyclohexylamine. To a solution of2-chloro-N-(5,6-dihydro-6-oxo-2-phenanthridinyl)-acetamide (45 mg) in N,N-dimethylfonnamide (10 mL) was added potassium phosphate (68 mg) andN-methylcyclohexylamine (0.025 mL). The solution stirred at roomtemperature for 5 hours followed by evaporation of the solvent. Thelight brown product was washed with water and filtered (30 mg). ¹H-NMR(300 MHz, DMSO-d₆), 11.69 (br s, 1 H), 9.76 (br s, 1 H), 8.65 (s, 1 H),8.34 (t, 2 H), 7.87 (m, 2 H), 7.66 (t, 1 H), 7.32 (t, 1 H), 3.18 (s, 2H), 2.34 (s, 3 H), 1.80 (m, 4 H), 1.60 (m, 2 H), 1.23 (m, 5 H).

EXAMPLE 9

[0141]

[0142] N-(5,6-dihydro-6-oxo-2-phenanthridinyl)-4-morpholineacetamide

[0143] Prepared from the compound of Example 2 and morpholine. To asolution of 2-chloro-N-(5,6-dihydro-6-oxo-2-phenanthridinyl)-acetamide(45 mg) in N, N-dimethylformamide (10 mL) was added potassium phosphate(68 mg) and morpholine (0.017 mL). The solution stirred at roomtemperature for 5 hours followed by evaporation of the solvent. Thelight brown product was washed with water and filtered (30 mg). ¹H-NMR(300 MHz, DMSO-d₆), 11.68 (br s, 1 H), 9.85 (br s, 1 H), 8.64 (s, 1 H),8.33 (d, J=9.0 Hz, 2 H), 7.89 (t, 1 H), 7.77 (d, J=9.0 Hz, 1 H), 7.66(t, 1 H), 7.32 (d, J=9.0 Hz, 1H), 3.68 (d, 4), 3.17 (s, 2 H), 2.55 (m,4H).

EXAMPLE 10

[0144]

[0145]5,6-Dihydro-N-[2-(1-piperidinyl)ethyl]-6-oxo-2-phenanthridinesulfonamidehydrogen chloride

[0146] Prepared from 2-(6(5 H)-phenanthridinone)sulfonyl chloride 4 and1-(2-aminoethyl)piperidine according to General Procedure A. A hydrogenchloride salt was formed upon adding 1 N HCl to a solution of5,6-dihydro-N-[2-(1-piperidinyl)ethyl]-6-oxo-2-phenanthridinesulfonamidein dioxane at 70° OC. The mixture was cooled down to 0° C produced asalt precipitation which was collected by filtration to give a whitesolid (53% yield from 4), mp 262-267° C. ¹H-NMR (400 MHz, CDCl₃), 8.79(s, 1 H), 8.56 (d, J=8.1 Hz, 1 H), 8.36 (d, J=7.2 Hz, 1 H), 7.93 (m, 2H), 7.75 (t, J=7.5 Hz, 1 H), 7.54 (d, J=8.7 Hz, 1 H), 3.41 (d, J=12.2Hz, 2 H), 3.14 (m, 4 H), 2.89 (t, 2 H). 1.71 (m, 1 H). Anal. (C₂₀ H₂₄ N₃O₃ S HCl 0.4 H₂O), C H N.

EXAMPLE 11

[0147]

[0148]5,6-Dihydro-N-[2-(4-morpholinyl)ethyl]-6-oxo-2-phenanthridinesulfonamide

[0149] Prepared from the compound 2-(6(5 H)-phenanthridinone)sulfonylchloride 4 and 1-(2-aminoethyl)morpholine according to General ProcedureA. Methylene chloride was used as solvent. Purification of theprecipitation by crystallization in dioxane gave a white solid (95%yield), mp 246-247° C. ¹H-NMR (400 MHz, CDCl₃), 12.04 (s, 2 H), 8.75 (s,1 H), 8.52 (d, J=8.2 Hz, 1 H), 8.35 (d, J=8.0 Hz, 1 H), 7.90 (m, 2 H),7.73 (t, J=7.5 Hz, 1 H), 7.50 (d, J=8.6 Hz, 1 H), 3.44 (t, J=4.5 Hz, 4H), 2.90 (t, J=6.1 Hz, 2 H), 2.30 (t, J=6.8 Hz, 2 H), 2.23 (t, J=4.3 Hz,4 H). Anal. (C₁₉ H₂₁ N₃ O₄ S 0.3 H₂O), C H N.

EXAMPLE 12

[0150]

[0151]N-[2-bis(2-Hydroxyethyl)amino]ethyl]-5,6-dihydro-6-oxo-2-phenanthridinesulfonamide

[0152] Prepared from the compound 2-(6(5 H-phenanthridinone)sulfonylchloride 4 and N,N-bis(2-hydroxyethyl)ethylenediamine according toGeneral Procedure A. Methylene chloride was used as solvent. Aprecipitation was formed after stirring the reactant for 20 hours.Purification of the precipitation by crystallization in p-dioxane gave ayellowish solid (40% yield), mp 210-214° C. (dec.). ¹H-NMR (400 MHz,DMSO-d₆), 8.72 (s, 1 H), 8.51 (d, J=8.0 Hz, 1 H), 8.35 (d, J=8.0 Hz, 1H), 7.92 (t, J=8.0 Hz, 1 H), 7.89 (d, J=4.0 Hz, 1 H), 7.73 (t, J=8.0 Hz,1 H), 7.51 (d, J=8.0Hz, 1 H), 4.34 (m, 2 H), 2.85 (m,2 H), 2.43 (t, 4H). Anal. (C₁₉ H₂₃ N₃ O₅ S 0.8 H₂O), C H N (: calcd 54.35 found 53.67).

EXAMPLE 13

[0153]

[0154] 3-(4-Morpholinylcarbonyl)-6(5 H)-phenanthridinone

[0155] Prepared from to 3-[6(5 H)Phenanthridinone]carbonyl chloride 7and morpholine according to General Procedure A. to 3-[6(5H)Phenanthridinone]carbonyl chloride 7 can be obtained by Schmidtinsertion of commercially available 2(fluoren-9-one)carboxylic acid asdescribed in the Scheme 3. Purification of compound by crystallizationin water-acetone gave a white solid (50 % yield), mp 182-184° C. ¹H-NMR(400 MHz, DMSO-d₆), 10.72(s, 1 H); 8.56(d, 1 H, 8.2 Hz); 8.52(d, 1 H,8.1 Hz); 8.34(dd, 1 H, 1.0 Hz, 7.9 Hz); 7.90(t, 1 H, 7.8 Hz); 7.69(t, 1H, 7.6 Hz); 7.47(d, 1 H, 7.4 Hz); 7.33(t, 1 H, 7.7 Hz); 3.84˜3.42(m, 8H). Anal. (C₁₈ H₁₆ N₂ O₃), C H N.

EXAMPLE 14

[0156]

[0157] N-[2-(4-Morpholinyl)ethyl] 5,6-dihydro-6-oxo-3-phenanthridinecarboxamide

[0158] Prepared from to 3-[6(5 H)Phenanthridinone]carbonyl chloride 7and 1-(2-aminoethyl)morpholine according to General Procedure A. DMF wasused as solvent. Purification of compound by crystallization in aceticacid gave a solid (60% yield), mp 285-292° C. ¹H-NMR (400 MHz, DMSO-d₆),11.82 (s, 1 H), 8.58 (d, 1 H, J=8 Hz), 8.54 (t 1 H, J=8 Hz), 8.34 (d, 1H, J=8Hz), 7.89 (t, 1 H, J=8 Hz), 7.83 (s, 1 H), 7.70 (m, 2 H), 3.58 (m,5 H), 3.40 (m, 2 H), 3.31 (m, 5 H). Anal. (C₂₀ H₂₁ N₃ O₃ 1Cl H), C H N.

EXAMPLE 15

[0159]

[0160] 1-[(5,6-Dihydro-6-oxo-3-phenanthridinyl)carbonyl]-proline

[0161] Prepared from to 3-[6(5 H)Phenanthridinone]-carbonyl chloride 7and (S)-methylproline in methylene chloride according to GeneralProcedure A. The resulting1-[(5,6-dihydro-6-oxo-3-phenanthridinyl)carbonyl]-proline methyl esterwas hydrolyzed with 1N sodium hydroxide in dioxane. After neutralizingthe mixture with HCl, the desired compound was collected as a whitesolid precipitate (60% yield), mp 250-253° C. ¹H-NMR (400 MHz, DMSO-d₆),12.60 (bs, 1 H), 11.79 (s, 1 H), 8.55 9d, 1 H, J=8 Hz), 8.47 (d, 1 H,J=8 Hz), 8.34 (d, 1 H, J=8 Hz), 7.90 (t, 1 H, J=8 Hz), 7.70 (t, 1 H,J=8Hz), 7.54 (s, 1 H), 7.40 (d, 1 H, J=8 Hz), 4.44 (m, 1 H), 3.56 (m, 2H), 2.37 (m,1 H), 1.90 (m, 3 H) Anal. (C₁₉ H₁₆ N₂ O₂), C H N.

EXAMPLE 16

[0162]

[0163] 8-Fluoro-N-[2-(4-morpholinyl)ethyl]5,6-dihydro-6-oxo-3-phenanthridinecarboxamide

[0164] Prepared from 3-[8-fluoro-6(5 H)phenanthridinone]carbonylchloride and 1-(2-aminoethyl)morpholine in dioxane according to GeneralProcedure A. 3-[8-fluoro-6(5h)phenanthridinone]carboxylic acid 6 wasprepared from Schimdt nitrogen insertion of 2(7-fluorofluoren-9-one)carboxylic acid as described in Scheme 3. Purification of theregioisomer mixture on silica gel column gave a desired isomer acid 6 asa solid. 3-[8-Fluoro-6(5 H)phenanthridinone]carboxylic acid sodium salthas ¹H-NMR (400 MHz, D₂O) 7.63 (m, 1 H), 7.47 (d, 1 H, J=8 Hz), 7.34 (m,2 H), 7.17 (m, 2 H), mp 300° C. (dec.). The acid 6 was further convertedto 3-[8-fluoro-6(5 H)phenanthridinone]carbonyl chloride using thionylchloride. After removal of the thionyl chloride, the residue was usedfor next step without purification.

[0165] Coupling reaction of 3-[8-fluoro-6(5 H)phenanthridinone]carbonylchloride and 1-(2-amninoethyl)morpholine afforded a brown solid, whichwas purified by crystallization in acetic acid. mp 285-290° C. ¹H-NMR(400 MHz, DMSO-d₆), 11.98 (s, 1 H), 8.67 (m, 1 H), 8.55 (t, 1 H, J=8Hz), 8.47 (d, 1 H, J=12 Hz), 7.99 (d, 1 H, J=8 Hz), 7.84 (s, 1 H), 7.79(t, 1 H, J=8 Hz), 7.70 (d, 1 H, J=8 Hz), 3.59 (m, 5 H), 3.41(m, 2 H),2.45 (m, 5 H). Anal. (C₂₀ H₂₀ F N₃ O₃), C H N (calcd 65.03 found 64.55).

EXAMPLE 17

[0166]

[0167]N-[(5,6-Dihydro-6-oxo-3-phenanthridinyl)carbonyl]-bis(benzyl)asparticacid ester

[0168] Prepared from to 3-[6(5 H)Phenanthridinone]carbonyl chloride 7and (S)-bisbezylaspartic acid ester in DMF according to GeneralProcedure A. The compound was purified by silica gel columnchromatography to give a white solid (70% yield), mp 162-164° C. ¹H-NMR(400 MHz, DMSO-d₆), 9.40 (dd, 1 H, J=7.22 Hz), 8.96 (S, 1 H), 8.66 (dd,1 H, J=8.08), 8.48 (dd, 1 H, J=8.08), 8.28 (dd, 1 H, J=10.3), 7.58 (t, 1H, J=7.22), 7.40 (m, 1 H). 7.22 (m, 10 H), 5.00 (m, 4 H), 4.85 (m, 1 H),3.15 (m, 2 H). Anal. (C₆₄ H₅₂ N₄ O₁₂), C H N.

EXAMPLE 18

[0169]

[0170] 8-Fluoro-3-[(4-methyl-1-piperazinyl)sulfonyl]- 6(5H)-phenanthridinone

[0171] Prepared from 3-[8-fluoro-6(5 H)phphenanthridinone]sulfonylchloride 6 and N-methylpiperazine in dioxane according to GeneralProcedure A. 3-[8-Fluoro-6(5 H)phenanthridinone]sulfonyl chloride 6 wasprepared in two steps.

[0172] Commercially available 2-fuloro-fluoren-9-one was placed in neatchorosulfonic acid under nitrogen at 0° C. The resulting mixture isallowed to warm to room temperature, continuously stirred for about 18hours and poured onto 100 g of ice. The residue is collected byfiltration and washed with water, ethanol to afford the product of7-(2-fuloro-fluoren-9-one)sulfonyl chloride as solid. The solid wasdirectly used for the next step without further purification.

[0173] A mixture of 7-(2-fuloro-fluoren-9-one)sulfonyl chloride (0.25 g)and sodium azide (0.25 g) in c-sulfric acid (10 mL) was stirred at 0° C.for 30 minutes and at 25° C. for 10 hours, and then poured into ice-coldwater (100 mL). A precipitation appeared upon adjusting the solution topH 8 with sodium hydroxide. The precipitation, a regioisomer mixture of3-[8-fluoro-6(5 H)phenanthridinone]sulfonyl chloride and of8-[3-fluoro-6(5 H)phenanthridinone]sulfonyl chloride, was collected byfiltration. The filtrate was allowed to stand at 0° C. for 24 hours, apure isomer of 3-[8-fluoro-6(5 H)phenantlridinone]sulfonyl chloride wasappeared as precipitate of a white crystal, ¹H-NMR (400 MHz, DMSO-d₆),400 MHz, 11.87(s, 1 H); 8.59(dd, 1 H, J=8.96 Hz, 5.04 Hz); 8.33(d, 1 H,J=8.4 Hz); 7.97(dd, 1 H, J=9.68 Hz, 2.96 Hz); 7.75(t, 1 H, J=8.6 Hz);7.68(s, 1 H); 7.47(d, 1 H, J=7.76).

[0174] Coupling of 3-[8-fluoro-6(5 H)phenanthridinone]sulfonyl chlorideand N-methylpiperazine in dioxane according to General Procedure Aprovided the final sulfonamide as a white solid, mp 260-261° C. (dec.).¹H-NMR (400 MHz, DMSO-d₆), 400 MHz 12.05(s, 1 H); 8.72(dd, 1 H, J 8 Hz,4 Hz); 8.65(d, 1 H, J=8 Hz); 8.02(dd, 1 H, J=8 Hz, 4 Hz); 7.83(td, 1 H,J=8 Hz, 4 Hz); 7.78(s, 1 H); 7.54(d, 1 H, J=8 Hz); 2.96(s, 4 H); 2.37(s,4 H); 2.13 (s, 3 H). Anal. (C₁₈ H₁₈ F N₃ O₃ S 0.3 H₂O), C H N.

EXAMPLE 19

[0175]

[0176] 8-Fluoro-N-[2-(4-morpholinyl)ethyl]5,6-dihydro-6-oxo-3-phenanthridinesulfonamide

[0177] Prepared from 3-[8-fluoro-6(5 H)phenanthridinone]sulfonylchloride 6 (see Example 13) and 1-(2-aminoethyl)morpholine in dioxaneaccording to General Procedure A. Purification of the crude byrecrystalization in acetic acid gave a white solid, the mp 240-241° C.¹H-NMR (400 MHz, DMSO-d₆), 12.08(s, 1 H); 8.70(dd, 1 H, J=8 Hz, 4 Hz);8.61(d, 1 H J=8 Hz); 8.01(dd, 1 H, J=8 Hz, 4 Hz); 7.83-7.73(m, 3 H);7.64(dd, 1 H, J=8 Hz, 4 Hz); 3.47(t, 4 H, J=4 Hz); 2.92(t, 2 H, J=8 Hz);2.31(t, 2 H, J=8 Hz); 2.26(t, 4 H, J=4 Hz). Anal. (C₁₉ H₂₀ F N₃ O₄SH₂O), C H N.

[0178] Other manners, variations or sequences of preparing the compoundsof the present invention will be readily apparent to those of ordinaryskill in the art.

[0179] The compounds of the present invention may be useful in the freebase form, in the form of base salts where possible, and in the form ofaddition salts, as well as in the free acid form. All these forms arewithin the scope of this invention. In practice, use of the salt formamounts to use of the base form. Pharmaceutically acceptable saltswithin the scope of this invention are those derived from mineral acidssuch as hydrochloric acid and sulfuric acid; and organic acids such asethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, andthe like, giving the hydrochloride, sulfonate, ethanesulfonate,benzenesulfonate, p-toluenesulfonate, and the like respectively, orthose derived from bases such as suitable organic and inorganic bases.Examples of pharmaceutically acceptable base addition salts withcompounds of the present invention include organic bases which arenontoxic and strong enough to form such salts. These organic bases andthe use thereof are readily understood by those skilled in the art.Merely for the purpose of illustration, such organic bases may includemono-, di-, and trialkylamines, such as methylamine, diethylamine andtriethylamine; mono-, di-, or trihydroxyalkylamines such as mono-, di-,and triethanolamine; amino acids such as arginine, and lysine;guanidine; N-methylglucosamine; N-methylgiucamine; L-glutamine;N-methylpiperazine; morpholine; ethylenedianane; N-benzylphenethylamine;tris(hydroxymethyl)antinoethane; and the like.

[0180] The acid addition salts of the basic compounds may he prepared bydissolving the free base of the compounds of the present invention inaqueous or aqueous alcohol solution or other suitable solventscontaining the appropriate acid or base and isolating the salt byevaporating the solution, or by reacting the free base of a compound ofthe present invention with an acid as well as reacting a compound of thepresent invention having an acid group thereon with a base such that thereactions are in an organic solvent, in which case the salt separatesdirectly or can be obtained by concentration of the solution.

[0181] The compounds of this invention contain one or more asymmetriccarbon atoms. Therefore, the invention includes the individualstereoisomers and mixtures thereof as well as the racemic compounds. Theindividual isomers may be prepared or isolated by methods known in theart.

[0182] The compounds of the invention exhibit pharmacological activityand are, therefore, useful as pharmaceuticals. In particular thecompounds exhibit central nervous and cardiac vesicular system activity.

[0183] Other variations and modifications of this invention using thesynthetic pathways described above will be obvious to those of ordinaryskill in the art.

[0184] Methods of Using the Compounds of the Invention

[0185] The compounds of the present invention can treat or preventtissue damage resulting from cell damage or death due to necrosis orapoptosis; can ameliorate neural or cardiovascular tissue damage,including that following focal ischemia, myocardial infarction, andreperfusion injury; can treat various diseases and conditions caused orexacerbated by PARP activity; can extend or increase the lifespan orproliferative capacity of cells; can alter the gene expression ofsenescent cells; and can radiosensitize cells. Generally, inhibition ofPARP activity spares the cells from energy loss, preventing, in the caseof neural cells, irreversible depolarization of the neurons, and thus,provides neuroprotection. While not being bound to any one particulartheory, it is thought that PARP activation may play a common role instill other excitotoxic mechanisms, perhaps as yet undiscovered, inaddition to the production of free radicals and NO.

[0186] For the foregoing reasons, the present invention further relatesto a method of administering a therapeutically effective amount of theabove-identified compounds in an amount sufficient to inhibit PARPactivity, to treat or prevent tissue damage resulting from cell damageor death due to necrosis or apoptosis, to effect a neuronal activity notmediated by NMDA toxicity, to effect a neuronal activity mediated byNMDA toxicity, to treat neural tissue damage resulting from ischemia andreperfusion injury, neurological disorders and neurodegenerativediseases; to prevent or treat vascular stroke; to treat or preventcardiovascular disorders; to treat other conditions and/or disorderssuch as age-related muscular degeneration, AIDS and other immunesenescence diseases, inflammation, gout, arthritis, atherosclerosis,cachexia, cancer, degenerative diseases of skeletal muscle involvingreplicative senescence, hyperglycemia, diabetes, head trauma, immunesenescence, inflammation, gout, inflammatory bowel disorders (such ascolitis and Crohn's disease), muscular dystrophy, osteoarthritis,osteoporosis, chronic and/or acute pain (such as neuropathic pain),renal failure, retinal ischemia, septic shock (such as endotoxic shock),and skin aging; to extend the lifespan and proliferative capacity ofcells; to alter gene expression of senescent cells; or to radiosensitizehypoxic tumor cells. The present invention also relates to treatingdiseases and conditions in an animal which comprises administering tosaid animal a therapeutically effective amount of the above-identifiedcompounds.

[0187] In particular, the present invention relates to a method oftreating, preventing or inhibiting a neurological disorder in an animal,which comprises administering to said animal a therapeutically effectiveamount of the above-identified compounds. In a particularly preferredembodiment, the neurological disorder is selected from the groupconsisting of peripheral neuropathy caused by physical injury or diseasestate, traumatic brain injury, physical damage to the spinal cord,stroke associated with brain damage, focal ischemia, global ischemia,reperfusion injury, demyelinating disease and neurological disorderrelating to neurodegeneration. Another preferred embodiment is when thereperfusion injury is a vascular stroke. Yet another preferredembodiment is when the peripheral neuropathy is caused by Guillain-Barresyndrome. Still another preferred embodiment is when the demyelinatingdisease and neurological disorder relates to neurodegeneration. Anotherpreferred embodiment is when the reperfusion injury is a vascularstroke. Still another preferred embodiment is when the demyelinatingdisease is multiple sclerosis. Another preferred embodiment is when theneurological disorder relating to neurodegeneration is selected from thegroup consisting of Alzheimer's Disease, Parkinson's Disease, andamyotrophic lateral sclerosis.

[0188] Yet another preferred embodiment is a method of treating,preventing or inhibiting a cardiovascular disease in an animal, such asangina pectoris, myocardial infarction, cardiovascular ischenia, andcardiovascular tissue damage related to PARP activation, byadministering to said animal an effective amount of the compounds of thepresent invention.

[0189] The present invention also contemplates the use of compound I,II, III, IV or V for inhibiting PARP activity, for treating, preventingor inhibiting tissue damage resulting from cell damage or death due tonecrosis or apoptosis, for treating, preventing or inhibiting aneurological disorder in an animal.

[0190] In a particularly preferred embodiment, the neurological disorderis selected from the group consisting of peripheral neuropathy caused byphysical injury or disease state, traumatic brain injury, physicaldamage to the spinal cord, stroke associated with brain damage, focalischemia, global ischemia, reperfusion injury, demyelinating disease andneurological disorder relating to neurodegeneration.

[0191] Another preferred embodiment is when the reperfiusion injury is avascular stroke. Yet another preferred embodiment is when the peripheralneuropathy is caused by Guillain-Barre syndrome. Still another preferredembodiment is when the demyelinating disease is multiple sclerosis.Another preferred embodiment is when the neurological disorder relatingto neurodegeneration is selected from the group consisting ofAlzheimer's Disease, Parkinson's Disease, and amyotrophic lateralsclerosis.

[0192] The present invention also contemplates the use of compound I,II, III, IV or V in the preparation of a medicament for the treatment ofany of the diseases and disorders in an animal described herein.

[0193] In a particular embodiment, the disease or disorder is aneurological disorder.

[0194] In a particularly preferred embodiment, the neurological disorderis selected from the group consisting of peripheral neuropathy caused byphysical injury or disease state, traumatic brain injury, physicaldamage to the spinal cord, stroke associated with brain damage, focalischemia, global ischemia, reperfusion injury, demyelinating disease andneurological disorder relating to neurodegeneration. Another preferredembodiment is when the reperfusion injury is a vascular stroke. Yetanother preferred embodiment is when the peripheral neuropathy is causedby Guillain-Barre syndrome.

[0195] Still another preferred embodiment is when the demyelinatingdisease is multiple sclerosis. Another preferred embodiment is when theneurological disorder relating to neurodegeneration is selected from thegroup consisting of Alzheimer's Disease, Parkinson's Disease, andamyotrophic lateral sclerosis.

[0196] The term “preventing neurodegeneration” includes the ability toprevent neurodegeneration in patients newly diagnosed as having aneurodegenerative disease, or at risk of developing a new degenerativedisease and for preventing further neurodegeneration in patients who arealready suffering from or have symptoms of a neurodegenerative disease.

[0197] The term “treatment” as used herein covers any treatment of adisease and/or condition in an animal, particularly a human, andincludes:

[0198] (i) preventing a disease and/or condition from occurring in asubject which may be predisposed to the disease and/or condition but hasnot yet been diagnosed as having it;

[0199] (ii) inhibiting the disease and/or condition, i.e., arresting itsdevelopment; or

[0200] (iii) relieving the disease and/or condition, i.e., causingregression of the disease and/or condition.

[0201] As used herein, the term “neural tissue damage resulting fromischemia and reperfusion injury” includes neurotoxicity, such as seen invascular stroke and global and focal ischemia. As used herein, the term“neurodegenerative diseases,” includes Alzheimer's disease, Parkinson'sdisease and Huntington's disease.

[0202] The term “ischemia” relates to localized tissue anemia due toobstruction of the inflow of arterial blood. Global ischemia occursunder conditions in which blood flow to the entire brain ceases for aperiod of time, such as may result from cardiac arrest. Focal ischemiaoccurs under conditions in which a portion of the brain is deprived ofits normal blood supply, such as may result from thromboembolyticocclusion of a cerebral vessel, traumatic head injury, edema, and braintumors.

[0203] The term “cardiovascular disease” relates to myocardialinfarction, angina pectoris, vascular or myocardial ischemia, andrelated conditions as would be known by those of skill in the art whichinvolve dysfunction of or tissue damage to the heart or vasculature, andespecially, but not limited to, tissue damage related to PARPactivation.

[0204] The term “radiosensitizer”, as used herein, is defined as amolecule, preferably a low molecular weight molecule, administered toanimals in therapeutically effective amounts to increase the sensitivityof the cells to be radiosensitized to electromagnetic radiation and/orto promote the treatment of diseases which are treatable withelectromagnetic radiation. Diseases which are treatable withelectromagnetic radiation include neoplastic diseases, benign andmalignant tumors, and cancerous cells. Electromagnetic radiationtreatment of other diseases not listed herein are also contemplated bythe present invention. The terms “electromagnetic radiation” and“radiation” as used herein includes, but is not limited to, radiationhaving the wavelength of 10⁻²⁰to 10⁰meters. Preferred embodiments of thepresent invention employ the electromagnetic radiation of:gamma-radiation (10⁻²⁰ to 10⁻¹³ m) x-ray radiation (10⁻¹¹ to 10⁻⁹ m),ultraviolet light (10 nm to 400 nm), visible light (400 nm to 700 nm),infrared radiation (700 nm to 1.0 mm), or microwave radiation (1 mm to30 cm).

[0205] Compositions and Methods for Effecting Neuronal Activity

[0206] Preferably, the compounds of the invention inhibit PARP activityand, thus, are believed to be useful for treating neural tissue damage,particularly damage resulting from cerebral ischemia and reperfusioninjury or neurodegenerative diseases in animals. The term “nervoustissue” refers to the various components that make up the nervous systemincluding, without limitation, neurons, neural support cells, glia,Schwann cells, vasculature contained within and supplying thesestructures, the central nervous system, the brain, the brain stem, thespinal cord, the junction of the central nervous system with theperipheral nervous system, the peripheral nervous system, and alliedstructures.

[0207] Further, according to the invention, an effective therapeuticamount of the compounds and compositions described above areadministered to animals to effect a neuronal activity, particularly onethat is not mediated by NMDA nearotoxicity. Such neuronal activity mayconsist of stimulation of damaged neurons, promotion of neuronalregeneration, prevention of neurodegeneration and treatment of aneurological disorder. Accordingly, the present invention furtherrelates to a method of effecting a neuronal activity in an animal,comprising administering an effective amount of the compound of formulaI, II, III, IV or V to said animal.

[0208] Examples of neurological disorders that are treatable by themethod of using the present invention include, without limitation,trigeminal neuralgia; glossopharyngeal neuralgia; Bell's Palsy;myasthenia gravis; muscular dystrophy; amyotrophic lateral sclerosis;progressive muscular atrophy; progressive bulbar inherited muscularatrophy; herniated, ruptured or prolapsed invertebrate disk syndromes;cervical spondylosis; plexus disorders; thoracic outlet destructionsyndromes; peripheral neuropathies such as those caused by lead,dapsone, ticks, porphyria, or Guillain-Barre syndrome; Alzheimer'sdisease; Huntington's Disease and Parkinson's disease.

[0209] The method of the present invention is particularly useful fortreating a neurological disorder selected from the group consisting of:peripheral neuropathy caused by physical injury or disease state; headtrauma, such as traumatic brain injury; physical damage to the spinalcord; stroke associated with brain damage, such as vascular strokeassociated with hypoxia and brain damage, focal cerebral ischemia,global cerebral ischemia, and cerebral reperfusion injury; demyelinatingdiseases, such as multiple sclerosis; and neurological disorders relatedto neurodegeneration, such as Alzheimer's Disease, Parkinson's Disease,Huntington's Disease and amyotrophic lateral sclerosis (ALS).

[0210] Treating Other PARP-Related Disorders

[0211] The compounds, compositions and methods of the present inventionare particularly useful for treating or preventing tissue damageresulting from cell death or damage due to necrosis or apoptosis.

[0212] The compounds, compositions and methods of the invention can alsobe used to treat a cardiovascular disorder in an animal, byadministering an effective amount of the compound of formula to theanimal. As used herein, the term “cardiovascular disorders” refers tothose disorders that can either cause ischemia or are caused byreperfusion of the heart. Examples include, but are not limited to,coronary artery disease, angina pectoris, myocardial infarction,cardiovascular tissue damage caused by cardiac arrest, cardiovasculartissue damage caused by cardiac bypass, cardiogenic shock, and relatedconditions that would be known by those of ordinary skill in the art orwhich involve dysfunction of or tissue damage to the heart orvasculature, especially, but not limited to, tissue damage related toPARP activation.

[0213] For example, the methods of the invention are believed to beuseful for treating cardiac tissue damage, particularly damage resultingfrom cardiac ischemia or caused by reperfusion injury in animals. Themethods of the invention are particularly useful for treatingcardiovascular disorders selected from the group consisting of: coronaryartery disease, such as atherosclerosis; angina pectoris; myocardialinfarction; myocardial ischemia and cardiac arrest; cardiac bypass; andcardiogenic shock. The methods of the invention are particularly helpfulin treating the acute forms of the above cardiovascular disorders.

[0214] Further, the methods of the invention can be used to treat tissuedamage resulting from cell damage or death due to necrosis or apoptosis,neural tissue damage resulting from ischenia and reperfusion injury,neurological disorders and neurodegenerative diseases; to prevent ortreat vascular stroke; to treat or prevent cardiovascular disorders; totreat other conditions and/or disorders such as age-related musculardegeneration, AIDS and other immune senescence diseases, inflammation,gout, arthritis, atherosclerosis, cachexia, cancer, degenerativediseases of skeletal muscle involving replicative senescence,hyperglycemia, diabetes, head trauma, immune senescence, inflammatorybowel disorders (such as colitis and Crohn's disease), musculardystrophy, osteoarthritis, osteoporosis, chronic and/or acute pain (suchas neuropathic pain), renal failure, retinal ischemia, septic shock(such as endotoxic shock), and skin aging; to extend the lifespan andproliferative capacity of cells; to alter gene expression of senescentcells; or to radiosensitize tumor cells

[0215] Further still, the methods of the invention can be used to treatcancer and to radiosensitize tumor cells. The term “cancer” isinterpreted broadly. The compounds of the present invention can be“anti-cancer agents”, which tern also encompasses “anti-tumor cellgrowth agents” and “anti-neoplastic agents”. For example, the methods ofthe invention are useful for treating cancers and radiosensitizing tumorcells in cancers such as ACTH producing tumors, acute lymphocyticleukemia, acute nonlymphocytic leukemia, cancer of the adrenal cortex,bladder cancer, brain cancer, breast cancer, cervical cancer, chroniclymphocytic leukemia, chronic myelocytic: leukemia, colorectal cancer,cutaneous T-cell lymphoma, endometrial cancer, esophageal cancer,Ewing's sarcoma, gallbladder cancer, hairy cell leukemia, head & neckcancer, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, livercancer, lung cancer (small and/or non-small cell), malignant peritonealeffusion, malignant pleural effusion, melanoma, mesothelioma, multiplemyeloma, neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, ovariancancer, ovary (germ cell) cancer, prostate cancer, pancreatic cancer,penile cancer, retinoblastoma, skin cancer, soft-tissue sarcoma,squamous cell carcinomas, stomach cancer, testicular cancer, thyroidcancer, trophoblastic neoplasms, uterine cancer, vaginal cancer, cancerof the vulva and Wilm's tumor.

[0216] The term “radiosensitizer”, as used herein, is defined as amolecule, preferably a low molecular weight molecule, administered toanimals in therapeutically effective amounts to increase the sensitivityof the cells to be radiosensitized to electromagnetic radiation and/orto promote the treatment of diseases which are treatable withelectromagnetic radiation. Diseases which are treatable withelectromagnetic radiation include neoplastic diseases, benign andmalignant tumors, and cancerous cells. Electromagnetic radiationtreatment of other diseases nct listed herein are also contemplated bythe present invention. The terms “electromagnetic radiation” and“radiation” as used herein includes, but is not limited to, radiationhaving the wavelength of 10⁻²⁰ to 10⁰ meters. Preferred embodiments ofthe present invention employ the electromagnetic radiation of:gamma-radiation (10⁻²⁰ to 10⁻¹³ m) x-ray radiation (10⁻¹¹ to 10⁻⁹ m),ultraviolet light (10 nm to 400 nm), visible light (400 nm to 700 mn),infrared radiation (700 mn to 1.0 mm), and microwave radiation (1 mm to30 cm).

[0217] Radiosensitizers are known to increase the sensitivity ofcancerous cells to the toxic effects of electromagnetic radiation.Several mechanisms for the mode of action of radiosensitizers have beensuggested in the literature including: hypoxic cell radiosensitizers (e.g., 2-nitroimidazole compounds, and benzotriazine dioxide compounds)promote the reoxygenation of hypoxic tissue and/or catalyze thegeneration of damaging oxygen radicals; non-hypoxic cellradiosensitizers (e.g., halogenated pyrimidines) can be analogs of DNAbases and preferentially incorporate into the DNA of cancer cells andthereby promote the radiation-induced breaking of DNA molecules and/orprevent the normal DNA repair mechanisms; and various other potentialmechanisms of action have been hypothesized for radiosensitizers in thetreatment of disease.

[0218] Many cancer treatment protocols currently employ radiosensitizersactivated by the electromagnetic radiation of x-rays. Examples of x-rayactivated radiosensitizers include, but are not limited to, thefollowing: metronidazole, misonidazole, desmethylmisonidazole,pimonidazole, etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233,EO9, RB 6145, nicotinamide, 5-bromodeoxyaridine (BUdR),5-iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine(FudR), hydroxyurea, cisplatin, and therapeutically effective analogsand derivatives of the same.

[0219] Photodynamic therapy (PDT) of cancers employs visible light asthe radiation activator of the sensitizing agent. Examples ofphotodynarnic radiosensitizers include the following, but are notlimited to: hematoporphyrin derivatives, Photofrin, benzoporphyrinderivatives, NPe6, tin etioporphyrin SnET2, pheoborbicle-a,bacteriochlorophyll-a, naphthalocyanines, phthalocyanines, zincphthalocyanine, and therapeutically effective analogs and derivatives ofthe same.

[0220] Radiosensitizers may be administered in conjunction with atherapeutically effective amount of one or more othei compounds,including but not limited to: compounds which promote the incorporationof radiosensitizers to the target cells; compounds which control theflow of therapeutics, nutrients, and/or oxygen to the target cells;chemotherapeutic agents which act on the tumor with or withoutadditional radiation; or other therapeutically effective compounds fortreating cancer or other disease. Examples of additional therapeuticagents that may be used in conjunction with radiosensitizers include,but are not limited to: 5-fluorouracil, leucovorin,5′-amino-5′deoxythymidine, oxygen, carbogen, red cell transfusions,perfluorocarbons (e.g., Fluosol-DA), 2,3-)PG, BW12C, calcium channelblockers, pentoxyfylline, antiangiogenesis compounds, hydralazine, andLBSO. Examples of chemotherapeutic agents that may be used inconjunction with radiosensitizers include, but are not limited to:adriamycin, camptothecin, carboplatin, cisplatin, daunorubicin,docetaxel, doxorubicin, interferon (alpha, beta, gamma), interleukin 2,irinotecan, paclitaxel, topotecan, and therapeutically effective analogsand derivatives of the same.

[0221] Pharmaceutical Compositions of the Invention

[0222] The present invention also relates to a pharmaceuticalcomposition comprising (i) a therapeutically effective amount of thecompound of formula I, II, III, IV or V and (ii) a pharmaceuticallyacceptable carrier.

[0223] The above discussion relating to the preferred embodiments'utility and administration of the compound of the present invention alsoapplies to the pharmaceutical composition of the present invention.

[0224] The term “pharmaceutically acceptable carrier” as used hereinrefers to any carrier, diluent, excipient, suspending agent, lubricatingagent adjuvant, vehicle, delivery system, emulsifier, disintegrant,absorbent, preservative, surfactant, colorant, flavorant, or sweetener.

[0225] For these purposes, the composition of the invention may beadministered orally, parenterally, by inhalation spray, adsorption,absorption, topically, rectally, nasally, bucally, vaginally,intraventricularly, via an implanted reservoir in dosage formulationscontaining conventional non-toxic pharmaceutically-acceptable carriers,or by any other convenient dosage form. The term parenteral as usedherein includes subcutaneous, intravenous, intramuscular,intraperitoneal, intrathecal, intraventricular, intrasternal, andintracranial injection or infusion techniques.

[0226] When administered parenterally, the composition will normally bein a unit dosage, sterile injectable form (solution, suspension oremulsion) which is preferably isotonic with the blood of the recipientwith a pharmaceutically acceptable carrier. Examples of such sterileinjectable forms are sterile injectable aqueous or oleaginous.suspensions. These suspensions may be formulated according to techniquesknown in the art using suitable dispersing or wetting agents andsuspending agents. The sterile injectable forms may also be sterileinjectable solutions or suspensions in non-toxic parenterally-acceptablediluents or solvents, for example, as solutions in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,saline, Ringer's solution, dextrose solution, isotonic sodium chloridesolution, and Hanks' solution. In addition, sterile, fixed oils areconventionally employed as solvents or suspending mediums. For thispurpose, any bland fixed oil may be employed including synthetic mono-or di-glycerides, corn, cottonseed, peanut, and sesame oil. Fatty acidssuch as ethyl oleate, isopropyl myristate, and oleic acid and itsglyceride derivatives, including olive oil and castor oil, especially intheir polyoxyethylated versions, are useful in the preparation ofinjectables. These oil solutions or suspensions may also containlong-chain alcohol diluents or dispersants.

[0227] Sterile saline is a preferred carrier, and the compounds areoften sufficiently water soluble to be made up as a solution for allforeseeable needs. The carrier may contain minor amounts of additives,such as substances that enhance solubility, isotonicity, and chemicalstability, e.g., anti-oxidants, buffers and preservatives.

[0228] Formulations suitable for nasal or buccal administration (such asself-propelling powder dispensing formulations) may comprise about 0.1%to about 5% w/w, for example 1% w/w of active ingredient. Theformulations for human medical use of the present invention comprise anactive ingredient in association with a pharmaceutically acceptablecarrier therefore and optionally other therapeutic ingredient(s).

[0229] When administered orally, the composition will usually beformulated into unit dosage forms such as tablets, cachets, powder,granules, beads, chewable lozenges, capsules, liquids, aqueoussuspensions or solutions, or similar dosage forms, using conventionalequipment and techniques known in the art. Such formulations typicallyirLclude a solid, semisolid, or liquid carrier. Exemplary carriersinclude lactose, dextrose, sucrose, sorbitol, mannitol, starches, gumacacia, calcium phosphate, mineral oil, cocoa butter, oil of theobroma,alginates, tragacanth, gelatin, syrup, methyl cellulose, polyoxyethylenesorbitan monolaurate, methyl hydroxybeizoate, propyl hydroxybenzoate,talc, magnesium stearate, and the like.

[0230] The composition of the invention is preferably administered as acapsule or tablet containing a single or divided dose of the inhibitor.Preferably, the composition is administered as a sterile solution,suspension, or emulsion, in a single or divided dose. Tablets maycontain carriers such as lactose and corn starch, and/or lubricatingagents such as magnesium stearate. Capsules may contain diluentsincluding lactose and dried corn starch.

[0231] A tablet may be made by compressing or molding the activeingredient optionally with one or more accessory ingredients. Compressedtablets may be prepared by compressing, in a suitable machine, theactive ingredient in a free-flowing form such as a powder or granules,optionally mixed with a binder, lubricant, inert diluent, su rfaceactive, or dispersing agent. Molded tablets may be made by molding in asuitable machine, a mixture of the powdered active ingredient and asuitable carrier moistened with an inert liquid diluent.

[0232] The compounds of this invention may also be administered rectallyin the form of suppositories. These compositions can be prepared bymixing the drug with a suitable non-irritating excipient which is solidat room temperature, but liquid at rectal temperature, and, therefore,will melt in the rectum to release the drug. Such materials includecocoa butter, beeswax, and polyethylene glycols.

[0233] Compositions and methods of the invention also may utilizecontrolled release technology. Thus, for example, the inventivecompounds may be incorporated into a hydrophobic polymer matrix forcontrolled release over a period of days. The composition of theinvention may then be molded into a solid implant, or externally appliedpatch, suitable for providing efficacious concentrations of the PARPinhibitors over a prolonged period of time without the need for frequentre-dosing. Such controlled release films are well known to the art.Particularly preferred are transdermal delivery systems. Other examplesof polymers commonly employed for this purpose that may be used in thepresent invention include nondegradable ethylene-vinyl acetate copolymeran degradable lactic acid-glycolic acid copolymers which may be usedexternally or internally. Certain hydrogels such aspoly(hydroxyethylmethacrylate) or poly(vinylalcohol) also may be useful,but for shorter release cycles than the other polymer release systems,such as those mentioned above.

[0234] In a preferred embodiment, the carrier is a solid biodegradablepolymer or mixture of biodegradable polymers with appropriate timerelease characteristics and release kinetics. The composition of theinvention may then be molded into a solid implant suitable for providingefficacious concentrations of the compounds of the invention over aprolonged period of time without the need for frequent re-dosing. Thecomposition of the present invention can be incorporated into thebiodegradable polymer or polymer mixture in any suitable manner known toone of ordinary skill in the art and may form a homogeneous matrix withthe biodegradable polymer, or may be encapsulated in some way within thepolymer, or may be molded into a solid implant.

[0235] It one embodiment, the biodegradable polymer or polymer mixtureis used to form a soft “depot” containing the pharmaceutical compositionof the present invention that can be administered as a flowable liquid,for example, by injection, but which remains sufficiently viscous tomaintain the pharmaceutical composition within the localized area aroundthe injection site. The degradation time of the depot so formed can bevaried from several days to a few years, depending upon the polymerselected and its molecular weight. By using a po ymer composition ininjectable form, even the need to make an incision may be eliminated. Inany event, a flexible or flowable delivery “depot” will adjust to theshape of the space it occupies with the body with a minimum of trauma tosurrounding tissues. The pharmaceutical composition of the presentinvention is used in amounts that are therapeutically effective, and maydepend upon the desired release profile, the concentration of thepharmaceutical composition required for the sensitizing effect, and thelength of time that the pharmaceutical composition has to be releasedfor treatment.

[0236] The PARP inhibitors are used in the composition in amounts thatare therapeutically effective. The compositions may be sterilized and/orcontain adjuvants, such as preserving, stabilizing, welling, oremulsifying agents, solution promoters, salts for regulating the osmoticpressure, and/or buffers. In addition, they may also contain othertherapeutically valuable substances. The compositions are preparedaccording to conventional mixing, granulating, or coating methods, andcontain about 0.1 to 75% by weight, preferably about 1 to 50% by weight,of the active ingredient.

[0237] To be effective therapeutically as central nervous systemtargets, the compounds of the present invention should readily penetratethe blood-brain barrier when peripherally administered. Compounds whichcannot penetrate the blood-brain barrier can be effectively administeredby an intraventricular route or other appropriate delivery systemsuitable for administration to the brain.

[0238] Doses of the compounds preferably include pharmaceutical dosageunits comprising an efficacious quantity of active compound. By anefficacious quantity is meant a quantity sufficient to inhibit PARP andderive its beneficial effects through administration of one or more ofthe pharmaceutical dosage units. Preferably, the dose is sufficient toprevent or reduce the effects of vascular stroke or otherneurodegenerative diseases.

[0239] For medical use, the amount required of the active ingredient toachieve a therapeutic effect will vary with the particular compound, theroute of administration, the mammal under treatment, and the particulardisorder or disease being treated. A suitable systematic dose of acompound of the present invention or a pharmacologically acceptable saltthereof for a mammal suffering from, or likely to suffer from, any ofcondition as described hereinbefore is in the range of about 0.1 mg/kgto about 100 mg/kg of the active ingredient compound the most preferreddosage being about 1 to about 10 mg/kg.

[0240] It is understood, however, that a specific dose level for anyparticular patient will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, rate of excretion,drug combination, and the severity of the particular disease beingtreated ancL form of administration.

[0241] It is understood that the ordinarily skilled physician orveterinarian will readily determine and prescribe the effective amountof the compound for prophylactic or therapeutic treatment of thecondition for which treatment i s administered. In so proceeding, thephysician or veterinarian could employ an intravenous bolus followed byan intravenous infusion and repeated administrations, parenterally ororally, as considered appropriate. While it is possible for an activeingredient to be administered alone, it is preferable to present it as aformulae on.

[0242] When preparing dosage form incorporating the compositions of theinvention, the compounds may also be blended with conventionalexcipients such as binders, including gelatin, pregelatinized starch,and the like; lubricants, such as hydrogenated vegetable oil, stearicacid, and the like; diluents, such as lactose, mannose, and sucrose;disintegrants, such as carboxymethylcellulose and sodium starchglycolate; suspending agents, such as povidone, polyvinyl alcohol, andthe like; absorbants, such as silicon dioxide; preservatives, such asmethylparaben, propylparaben, and sodium benzoate; surfactants, such assodium lautyl sulfate, polysorbate 80, and the like; colorants such asF.D.& C. dyes and lakes; flavorants; and sweeteners.

[0243] The present invention relates to the use of compounds I, II, III,IV or V in the preparation of a medicament for the treatment of anydisease or disorder in an animal described herein.

[0244] PARP Assays

[0245] IC₅₀

[0246] A convenient method to determine IC₅₀ of a PARP inhibitorcompound is a PARP assay using purified recombinant human PARP fromTrevigan (Gaithersburg, Md.), as follows: The PARP enzyme assay is setup on ice in a volume of 100 microliters consisting of 100 mM Tris-HCI(pH 8.0), 1 mM MgCI₂, 28 mM KCl, 28 mM NaCl, 0.1 mg/ml of DNase Iactivated herring sperm DNA (Sigma, Mo.), 3.0 micromolar [3 H]nicotinamide adenine dinucleotide (470 mci/mmole), 7 micrograms/ml PARPenzyme, and various concentrations of the compounds; to be tested. Thereaction is initiated by incubating the mixture at 25° C. After 15minutes of incubation. the reaction is terminated by adding 500microliters of ice cold 20% (w/v) trichloroacetic acid. The precipitateformed is transferred onto a glass fiber filter (Packard Unifilter-GF/B)and washed three times with ethanol. After the filter is dried, theradioactivity is determined by scintillation counting. The compounds ofthis invention found to have potent enzymatic activity in the range of afew nM to 20 μM in IC₅₀ in this inhibition assay.

[0247] Using the PARP assay described above, approximate IC₅₀ (μM)values obtained as shown in the following Table III: TABLE III CompoundNo. IC₅₀ (μM) 1 0.12 2 100 3 0.03 4 0.14 5 0.08 6 0.11 7 0.03 8 0.06 90.04 10 0.09 13 0.01 15 1.27 16 0.399 17 0.415 18 0.15 19 0.157 20 0.03321 0.124 22 0.115 23 0.01 24 0.056 25 0.272 26 0.711 27 0.111 28 0.34129 0.054 30 0.083 31 0.069 32 0.036 33 0.045 34 0.065 35 0.083 36 0.0237 0.088 38 0.031 39 0.025 40 0.035 41 0.118 42 0.019 43 0.108 44 0.01945 0.229 46 0.044 47 0.152 48 0.037 49 0.021 50 0.08 51 0.099 52 1.2 530.074 54 0.195 55 0.094 56 5.9 57 0.415 58 1.1 59 7.5 60 11.5 61 0.31962 0.449 63 0.174 64 0.043 65 0.2 66 0.099 67 0.164 68 0.142

[0248] Focal cerebral ischemia

[0249] The following focal cerebral ischemia assay is useful fordetermining the PARP inhibiting effects of the compounds of the presentinvention. The following examples demonstrate that compounds related tothose of the present invention are effective in inhibiting PARPactivity.

[0250] Focal cerebral ischemia is produced by cauterization of the rightdistal MCA (middle cerebral artery) with bilateral temporary commoncarotid artery occlusion in male Long-Evans rats for 90 minutes. Allprocedures performed on the animals are approved by the UniversityInstitutional Animal Care and Use Committee of the University ofPennsylvania. A total of 42 rats (weights: 230-340 g) obtained fromCharles River were used in this study. The animals fasted overnight withfree access to water prior to the surgical procedure

[0251] Two hours prior to MCA occlusion, varying amounts (control, n=14;5 mg/kg, n=7; 10 mg/kg, n=7; 20 mg/kg, n=7; and 40 mg/kg, n=7) of thecompound, 3,4-dihydro-5-[4-(1-piperidinyl)-butoxy]-1(2 H)-isoquinolinone(“DPQ”) were dissolved in dimethyl sulfoxide (DMSO) using a sonicator. Avolume of 1.28 ml/kg of the resulting solution was injectedintraperitoneally into fourteen rats.

[0252] The rats were then anesthetized with halothane (4% for inductionand 0.8%-1.2% for the surgical procedure) in a mixture of 70% nitrousoxide and 30% oxygen. The body temperature was monitored by a rectalprobe and maintained at 37.5+0.5° C. with a heating blanket regulated bya homeothermic blanket control unit (Harvard Apparatus Limited, Kent,U.K.). A catheter (PE-50) was placed into the tail artery, and arterialpressure was continuously monitored and recorded on a Grass polygraphrecorder (Model 7D, Grass Instruments, Quincy, Mass.). Samples for bloodgas analysis (arterial pH, PaO₂ and PaCO₂) were also taken from the tailartery catheter and measured with a blood gas analyzer (ABL 30,Radiometer, Copenhagen, Denmark). Arterial blood samples were obtained30 minutes after MCA occlusion.

[0253] The head of the animal was positioned in a stereotaxic frame, anda right parietal incision between the right lateral canthus and theexternal auditory meatus was made. Using a dental drill constantlycooled with saline, a 3 mm burr hole was prepared over the cortexsupplied by the right MCA, 4 mm lateral to the sagittal suture and 5 mmcaudal to the coronal suture. The dura mater and a thin inner bone layerwere kept, care being taken to position the probe over a tissue areadevoid of large blood vessels. The flow probe (tip diameter of 1 mm,fiber separation of 0.25 mm) was lowered to the bottom of the cranialburr hole using a micromanipulator. The probe was held stationary by aprobe holder secured to the skull with dental cement. The microvascularblood flow in the right parietal cortex was continuously monitored witha laser Doppler flowmeter (FloLab, Moor, Devon, U.K., and Periflux 4001,Perimed, Stockholm, Sweden).

[0254] Focal cerebral ischemia was produced by cauterization of thedistal portion of the right MCA with bilateral temporary common carotidartery (CCA) occlusion by the procedure of Chen et al., “A Model ofFocal Ischemic Stroke in the Rat: Reproducible Extensive CorticalInfarction”, Stroke 17:738-43 (1986) and/or Liu et al., “PolyethyleneGlycol-conjugated Superoxide Dismutase and Catalase Reduce IschemicBrain Injury”, Am. J. Physiol. 256:H589-93 (1989), both of which arehereby incorporated by reference.

[0255] Specifically, bilateral CCA's were isolated, and loops made frompolyethylene (PE-10) catheter were carefully passed around the CCA's forlater remote occlusion. The incision made previously for placement ofthe laser doppler probe was extended to allow observation of the rostralend of the zygomatic arch at the fusion point using a dental drill, andthe dura mater overlying the MCA was cut. The MCA distal to its crossingwith the inferior cerebral vein was lifted by a fine stainless steelhook attached to a micromanipulator and, following bilateral CCAocclusion, the MCA was cauterized with an electrocoagulator. The burrhole was covered with a small piece of Gelform, and the wound wassutured to maintain the brain temperature within the normal ornear-normal range.

[0256] After 90 minutes of occlusion, the carotid loops were released,the tail arterial catheter was removed, and all of the wounds weresutured. Gentamicin sulfate (10 mg/ml) was topically applied to thewounds to prevent infection. The anesthetic was discontinued, and theanimal was returned to his cage after awakening. Water and food wereallowed ad libitum.

[0257] Two hours after MCA occlusion, the animals were given the samedoses of the PARP inhibitor as in the pre-treatment. Twenty-four hoursafter MCA occlusion, the rats were sacrificed with an intraperitonealinjection of pentobarbital sodium (150 mg/kg). The brain was carefullyremoved from the skull and cooled in ice-cold artificial CSF for fiveminutes. The cooled brain was then sectioned in the coronal plane at 2mm intervals using a rodent brain matrix (RBM-4000C, ASI Instruments,Warren, Mich.). The brain slices were incubated in phosphate-bufferedsaline containing 2% 2,3,5-triphenyltetrazolium chloride (TTC) at 37° C.for ten minutes. Color photographs were taken of the posterior surfaceof the stained slices and were used to determine the damaged area ateach cross-sectional level using a computer-based image analyzer (NIHImage 1.59). To avoid artifa(ts due to edema, the damaged area wascalculated by subtracting the area of the normal tissue in thehemisphere ipsilateral to the stroke from the area of the hemispherecontralateral to the stroke, by the method of Swanson et al., “ASemiautomated Method for Measuring Brain Infarct Volume”, J. Cereb.Blood Flow Metabol. 10:290-93 (1990), the disclosure of which is herebyincorporated by reference. The total volume of infarction was calculatedby summation of the damaged volume of the brain slices.

[0258] The cauterization of the distal portion of the right MCA withbilateral temporary CCA occlusion consistently produced awell-recognized cortical infarct in the right MCA territory of each testanimal. There was an apparent uniformity in the distribution of thedamaged area as measured by TTC staining in each group, as shown in FIG.1.

[0259] In FIG. 1, the distribution of the cross-sectional infarct areaat representative levels along the rostrocaudal axis was measured fromthe interaural line in non-treated animals and in animals treated with10 mg/kg of 3,4-dihydro-5-[4-(1-piperidinyl)-butoxy]-1(2H)-isoquinolinone. The area of damage was expressed as mean+standarddeviation. Significant differences between the 10 mg-treated group andthe control group were indicated (⁸p<0.02, ^(**)p<0.01, ^(**)p<0.001).The 5 mg/kg and 20 mg/kg curves fell approximately halfway between thecontrol and the 10 mg/kg curves, whereas the 40 mg/kg curve was close tothe control. The 5, 20 and 40 mg/kg curves were omitted for clarity.

[0260] PARP inhibition led to a significant decrease in the damagedvolume in the 5 mg/kg-treated group (106.7+23.2 mm³, p<0.001), the 10mg/kg-treated group (76.4+16.8 mm, p<0.001), and the 20 mg/kg-treatedgroup (110.2+42.0 mm³, p<0.01), compared to the control group(165.2+34.0 mm³). The data are expressed as mean+standard deviation. Thesignificance of differences between groups was determined using ananalysis of variance (ANOVA) followed by Student's t-test for individualcomparisons.

[0261] There was no significant difference between the control and the40 mg/kg reated group (135.6+44.8 mm³). However, there were significantdifferences between the 5 mg/kg-treated group and the 10 mg/kg-treatedgroup (p<0.02), and between the 10 mg/kg-treated group and the 40mg/kg-treated group (p<0.01), as shown in FIG. 2.

[0262] In FIG. 2, the effect of intraperitoneal administration of3,4-dihydro-5-[4-(1-piperidinyl)-butoxy]-1 (2 H)-isoquinolinone on theinfarct volume was depicted graphically. The volumes of infarct wereexpressed as mean+standard deviation. Significant differences betweenthe treated groups and the control group were indicated (^(*)p<0.01,^(**)p<0.001). It is not clear why a high dose (40 mg/kg) of the PARPinhibitor, 3,4-dihydro-5-[4-(1-piperidinyl)-butoxy]-1(2H)-isoquinolinone, was less neuroprotective. The U-shaped dose-responsecurve may suggest dual effects of the compound.

[0263] However, overall, the in vivo administration of the inhibitor ledto a substantial reduction in infarct volume in the focal cerebralischemia model in the rat. This result indicated that the activation ofPARP plays an important role in the pathogenesis of brain damage incerebral ischemia.

[0264] The values of arterial blood gases (PaO₂, PaCO₂ and pH) werewithin the physiological range in the control and treated groups with nosignificant differences in these parameters among the five groups, asshown below in Table IV. A “steady state” MABP was taken followingcompletion of the surgical preparation, just prior to occlusion; an“ischemia” MABP was taken as the average MABP during occlusion. TABLE IVMABP (mm Hg) PaO₂ PaCO₂ Steady Ischemia (mm Hg) (mm Hg) pH State Control125 ± 21 38.6 ± 4.6 7.33 ± 0.05  79 ± 14 91 ± 13** group (n = 4) 5mg/kg- 126 ± 20 38.0 ± 2.8 7.36 ± 0.02 78 ± 5 91 ± 12** treated group (n= 7) 10 mg/kg- 125 ± 16 39.3 ± 5.2 7.34 ± 0.03 80 ± 9 90 ± 14*  treatedgroup (n = 7) 20 mg/kg- 122 ± 14 41.3 ± 2.8 7.35 ± 0.23  79 ± 10 91 ±12** treated group (n = 7) 40 mg/kg- 137 ± 17 39.5 ± 4.7 7.33 ± 0.24 78± 4 88 ± 12*  treated group (n = 7)

[0265] There were no significant differences in any physiologicalparameter, including mean arterial blood pressure (MABP), prior to MCAand CCA occlusion among the five groups. Although MABP was significantlyelevated following occlusion in all five groups, there were nosignificant differences in MABP during the occlusion period among thegroups.

[0266] Since the blood flow values obtained from the laser doppler werein arbitrary units, only percent changes from the baseline (prior toocclusion) were reported. Right MCA and bilateral CCA occlusion produceda significant decrease in relative blood flow in the right parietalcortex to 20.8+7.7% of the baseline in the control group (n=5),18.7+7.4% in the 5 mg/kg-treated group (n=7), 21.4+7.7% in the 10mg/kg-treated group (n=7) and 19.3+11.2% in the 40 mg/kg-treated group(n=7). There were no significant differences in the blood flow responseto occlusion among the four groups. In addition, blood flow showed nosignificant changes truoughout the entire occlusion period in any group.

[0267] Following release of the carotid occlusions, a good recovery ofblood flow (sometimes hyperemia) was observed in the right MCA territoryof all animals. Reperfusion of the ischemic tissue resulted in theformation of NO and peroxynitrite, in addition to oxygen-derived freeradicals. All of these radicals have been shown to cause DNA strandbreaks and to activate PARP.

[0268] This example provided evidence that the related compounds of thepresent invention are effective in inhibiting PARP activity.

[0269] Exemplified compounds of the present invention may be tested fortheir ability to reduce focal cerebral ischemia in the followingsimplified procedure. Rats are allowed free access to water and rat chow(Wayne, Chicago, Ill.) until surgery. Housing and anesthesia concur withguidelines established by the institutional Animal Studies Committee,and are in accordance with the PHS Guide for the Care and Use ofLaboratory Animals, USDA Regulations, and the AVMA Panel on Euthanasiaguidelines.

[0270] The animals are anesthetized with isofluorane (induction, 3%;maintenance, 1.25% in a mixture of 30% O₂ and 70% NO₂ through a facemask. The rectal temperature is maintained at 37° C. with a homeothermicblanket (Harvard Apparatus, South Natick, Mass.). First, an iv catheteris inserted into the left femoral vein and the line run up through thenape of the neck for connection to a tethered swivel (InstechLaboratories, Plymouth Meeting, PA) and remote infusion pump (StoeltingInc., Wood Dale, Ill.). In some cases, the right femoral artery iscannulated for monitoring arterial blood pressure and heart rate and forobtaining blood samples for arterial blood gas.

[0271] The right middle cerebral artery (MCA) is then exposed by makingvertical skin incision midway between the right eye and ear andoverlying skull is removed with a dental drill (Chen et al, 1986). Afterincision of the dura, the artery is coagulated at the level of theinferior cerebral vein with a bipolar cautery unit (Valleylab NS2000,Boulder, Colo.), and cut to prevent spontaneous reperfusion (Takahashiet al., 1997). Both common carotid arteries (CCAs) that had beenpreviously isolated and freed of soft tissues and nerves are thenligated using non-traumatic aneurysm clips. After the wounds are closedwith surgical clips, the animals are allowed to recover from anesthesiaand returned to their cage which is warmed to 27° C. with a heated waterunderpad and humidified warm tent.

[0272] The PARP inhibitor to be tested is first administered 30 minafter MCAO as an iv bolus, 10 mg/kg infused over 5 min, followed by a 12hr continuous infusion of 2 mg/kg/hr (0.3 ml/hr). Ninety minutes afterthe MCAO, the animals are removed from the infusion tether, brieflyreanesthetized with isofluorane, and the carotid clips are removed . Theanimal is returned to its warm cage and reconnected to the iv infusionpump for the duration of the experiment.

[0273] At 24 hrs after permanent MCAO, animals are sedated with ketamineand the heads removed by guillotine. Brains are removed, cooled inice-cold saline, and sliced into 2 mm coronal sections using a rat brainmatrice (Harvard Bioscience, South Natick, Mass.). The brain slices areincubated in phosphate-buffered saline (pH 7.4) containing 2% TTC at 37°C. for 10 min. and then stored in 10% neutral-buffered formalin.

[0274] Cross-sectional area of the TTC-unstained region for each brainslice is determined using an image analyzer (MetaMorph, UniversalImaging Corp., West Chester, Pa.). The total volume of infarction in theright hemisphere is calculated by summation of the direct (TTC-negative)and indirect measures of the infarct areas in the component brainslices. The infarcted volumes in vehicle and drug-treated groups (n=8)are tested for significant statistical difference using an unpairedStudent-t test.

[0275] Various doses of the compounds of the invention may be tested inthis model. The compounds are administered in either a single dose or aseries of multiple doses, i.p. or i.v., at different times, both beforeor after the onset of ischemia. Compounds of the invention are expectedto provide protection from ischemia in the range of about 0 to 80%.

[0276] Heart Ischenia/Reperfusion Injury

[0277] The experiments of the heart ischemia/reperfusion injury model isperformed using female Sprague-Dawley rats weighing 250-300 g which areanesthetized with sodium pentobarbital at dose of 65 mg/kgintraperitoneally. The rectal temperature is maintained at 37° C. byusing a homeothermic blanket system (Harvard Apparatus, South Natick,Mass.). The trachea is cannulated and the rat is ventilated with RoomAir by using Harvard Rodent Ventilator (Harvard Apparatus, South Natick,Mass.). The left jugular vein is cannulated with PE-50 tubing for drugdelivery. The right carotid artery is cannulated for BP measurement. Theheart is exposed by opening the chest at the 4^(th) left intercostalspace. A main left branch of coronary artery (LAD) is occluded by 4-0silk ligature for 30 min of ischemia and released for 90 min ofreperfusion. During the experiment, arterial BP and EKG are monitored byMicro-Med Cardiovascular System (Louisville, Ky.).

[0278] At the end of reperfusion, the LAD coronary artery is re-occludedand about 2 ml of 5% Evans Blue dye is injected through i.v. line todistinguish the ischemic area from non-ischemic area of the heart. Thenthe heart is immediately taken off and frozen in the freezer. After atleast 30 min of freezing, the heart is sliced into five sections with 2mm thickness and stained in 1% TTC solution for 30 min at 37° C., Theright ventricle is trimmed off. Infarct area, risk area and total leftventricular area in each face of the section are measured by using animage analysis system (BIOQUANT, Nashville, Tenn.). The infarct size iscalculated as the percent total infarct volume of the total risk volume.

[0279] For drug treated group, compounds are administered according tothe following three protocols: 1. Single dose of compound is given 60min prior to the onset of ischemia through i. p. injection. 2. Compoundis delivered through i.v. bolus 1 min before the onset of ischemiafollowed by i.v. infusion until the end of reperfusion. 3. Compound isdelivered through i.v. bolus 1 min before the onset of reperfusionfollowed by i.v. infusion until the end of reperfusion. For eachdrug-treated group, there is a corresponding vehicle treated group withn=6 or n=8. The difference between vehicle and drug treated groups iscompared by using unpaired t-test with p<0.05. Various doses ofcompounds are tested in this model. The compounds are given in eithersingle or multiple doses, i.p or i.v., at different times before orafter the onset of ischemia. The compounds of this invention areexpected to have ischemia/reperfusion injury protection in the range of10 to 40 percent in this assay.

[0280] As a result of the PARP inhibition activity, the compounds ofthis invention are expected to protect against ischemia-induceddegeneration of rat cortical neurons in vitro and thus may be useful indisorders arising from cerebral ischemia such as stroke, septic shock,or CNS degenerative disorders. They may also be useful in protecting thespinal cord following trauma. As an experimental result ofischemialreperfusion injury in rats, the present invention is furtherdirected to a method of prophylactic or therapeutic treatment of heartattack, cardiac arrest, cardiac bypass, hyperglycemia, diabetes, or riskof damage which comprises administering an effective amount of acompound of the present invention for PARP inhibition in unit dosageform.

[0281] In vitro Radiosensitization

[0282] In vitro radiosensitization may be measured with the use of ahuman prostate cancer cell line, PC-3s, which are plated in 6 welldishes and grown at monolayer cultures in RPMI1640 supplemented with 10%FCS. The cells are maintained at 37° C. in 5% CO₂ and 95% air. The cellsare exposed to a dose response (0.1 mM to 0.1 μM) of 3 different PARPinhibitors prior to irradiation at one sublethal dose level. For alltreatment groups, the six well plates are exposed at room temperature ina Seifert 250 kV/15mA irradiator with a 0.5 mm Cu/l nun. Cell viabilityis examined by exclusion of 0.4% trypan blue. Dye exclusion is assessedvisually by microscopy and viable cell number was calculated bysubtracting the number of cells from the viable cell number and dividingby the total number of cells. Cell proliferation rates are calculated bythe amount of ³H-thymidine incorporation post-irradiation. The PARPinhibitors are expected to radiosensitize the cells.

[0283] Measuring Altered Gene Expression in mRNA Senescent Cells

[0284] Gene expression alteration may be measured with human fibroblastBJ cells which, at Population Doubling (PDL) 94, are plated in regulargrowth medium and then changed to low serum medium to reflectphysiological conditions described in Linskens, et al., Nucleic AcidsRes. 23:16:3244-3251 (1995). A medium of DMEM/199 supplemented with 0.5%bovine calf serum is used. The cells are treated daily for 13 days. Thecontrol cells are treated with and without the solvent used toadminister the PARP inhibitor. The untreated old and young control cellsare tested for comparison. RNA is prepared from the treated and controlcells according to the techniques described in PCT Publication No.96/13610 and Northern blotting is conducted. Probes specific forsenescence-related genes are analyzed, and treated and control cellscompared. In analyzing the results, the lowest level of gene expressionis arbitrarily set at 1 to provide a basis for comparison. Three genesparticularly relevant to age-related changes in the skin are collagen,collagenase and elastin. West, Arch. Derm. 130:87-95 (1994). Elastinexpression of the cells treated with the PARP inhibitor is expected tobe significantly increased in comparison with the control cells. Elastinexpression should be significantly higher in young cells compared tosenescent cells, and thus treatment with the PARP inhibitor should causeelastin expression levels in senescent cells to change to levels similarto those found in much younger cells. Similarly, a beneficial effectshould be seen in collagenase and collagen expression with treatmentwith the PARP inhibitors.

[0285] Neuroprotective Effects of PARP Inhibitors on ChronicConstriction Injury (CCI) in Rats Adult male Sprague-Dawley rats,300-350 g, are anesthetized with intraperitoneal 50 mg/kg sodiumpentobarbital. Nerve ligation is performed by exposing one side of therat's sciatic nerves and dissecting a 5-7 mm-long nerve segment andclosing with four loose ligatures at a 1.0-1.5-mm, followed byimplanting of an intrathecal catheter and inserting of a gentamicinsulfate-flushed polyethylene (PE-10) tube into the subarachnoid spacethrough an incision at the cisterna magna. The caudal end of thecatheter is gently threaded to the lumbar enlargement and the rostralend is secured with dental cement to a screw embedded in the skull andthe skin wound is closed with wound clips.

[0286] Thermal hyperalgesia to radiant heat is assessed by using apaw-withdrawal test. The rat is placed in a plastic cylinder on a 3-mmthick glass plate with a radiant heat source from a projection bulbplaced directly under the plantar surface of the rat's hindpaw. Thepaw-withdrawal latency is defined as the time elapsed from the onset ofradiant heat stimulation to withdrawal of the rat's hindpaw.

[0287] Mechanical hyperalgesia is assessed by placing the rat in a cagewith a bottom made of perforated metal sheet with many small squareholes. Duration of paw-withdrawal is recorded after pricking themid-plantar surface of the rat's hindpaw with the tip of a safety pininserted through the cage bottom.

[0288] Mechano-allodynia is assessed by placing a rat in a cage similarto the previous test, and applying von Frey filaments in ascending orderof bending force ranging from 0.07 to 76 g to the mid-plantar surface ofthe rat's hindpaw. A von Frey filament is applied perpendicular to theskin and depressed slowly until it bends. A threshold force of responseis defined as the first filament in the series to evoke at least oneclear paw-withdrawal out of five applications.

[0289] Dark neurons are observed bilaterally within the spinal corddorsal horn, particularly in laminae I-II, of rats 8 days afterunilateral sciatic nerve ligation as compared with sham operated rats.Various doses of PARP inhibitors are tested in this model and shown toreduce both incidence of dark neurons and neuropathic pain behavior inCCI rats.

[0290] Treatment and Prevention of Gout and Symptoms of Gout

[0291] Deposition of crystals of monosodium urate (MSU crystals) in thejoint articular space is the etiological cause of inflammatorypathologies such as gout and pseudogout. Clinically, these inflammatorydiseases are associated with oedema and erythema of the joints withconsequently severe pain. A strong infiltration of leucocytes in theintraarticular and periarticular space leading to: 1) acute, episodicarticular and periarticular inflammation, and 2) chronic articularchanges, are also characteristic of this pathology. It has long beenclear that neutrophils are the predominant cell type recovered fromthese inflammatory joints (Dieppe et al., (1979). Synovial fluidcrystals. Q. J. Med. XLVIII: 533-553; Terkletaub, (1991).Monocyte-derived neutrophil chemotactic factor/interleukin-8 is apotential mediator of crystal-induced inflammation. Arth. Rheum. 34:894-903.). A better understanding of the inflammatory processes elicitedby MSU crystals, and the fact that there is a clear relationship betweenthese crystals and gouty arthritis, has prompted the characterisation ofexperimental models of crystal-induced inflammation. Examples of modelswhere crystal challenge has led to cell recruitment into specificcavities, are canine joints (Phelps & McCarty, 1966, Ann Int. Med. 9:115-125), rat pleurisy (Deporter et al., 1979, Br. J. PharmacoL 65:163-165; Sedgwick et al., 1985, Agents Actions 17: 209-213), andutilisation of a pre-formed rat air-pouch (Brookes et al., 1987). Thelatter experimental system has shown that neutrophil accumulation wasrelated to generation of chemoattractants such as LTB4, which wassubsequently inhibited by colchicine (Brooks et aL, 1987, Br. J.Pharmacol. 90: 413-419).

[0292] Neutrophils have been shown to be activated by MSU crystals,releasing an array of mediators that may be, in part, responsible forthe local and systemic inflammatory manifestations found incrystal-induced joint disorders. The crystals interact with neutrophilsleading to the release of lysomal enzymes (Hoffstein et al., 1975, Arth.Rheum. 18: 153-165), release of oxygen derived free radicals (Simchowitzet al., 1982, Arth. Rheum. 25: 181-188; Abramson et aL, 1982, ArthrRheum. 25: 174-180), induction of phospholipase A₂ (PLA₂) in leucocytes(Bomalaski et al, 1990, J. Immunol. 145: 3391-3397), and activation ofsynthesis of 5-lipoxygenase products (Poubelle et al., 1987, Biochem.Biophys. Res. Commun. 149: 649-657).

[0293] In vitro, MSU crystals have been shown to release the cytokineinterleukin-1β(IL-1β) from human neutrophils, adding this stimulus to alist of others that also release this cytokine, such as zymosan, LPS,phorbol esters, granulocyte macrophage-colony stimulating hormone(GM-CSF) and TNF-alpha. Furthermore it has also been shown that humanmonocytes and synoviocytes can synthesise and release various cytokinessuch as IL-6 and IL-8 (Guerne et al., 1989, Arth. Rheum. 32: 1443-1452;Terkeltaub et al., 1991, Arth. Rheum. 34: 894-903). In addition,colchicine selectively inhibits MSU crystal- and TNF-□ induced releaseof IL-1β(Roberge et aL, 1994, J. Immunol. 152: 5485-5494).

[0294] In experimental models of gout the synthesis of a CXC chemokineselective for neutrophils, such as IL-8, has also been observed, but notthat of a CC chemokine monocyte chemoattractant protein-1(MCP-1)(Hachicha et al., 1995, J. Exp. Med. 182: 2019-2025). These resultssuggest that production of IL-8 and abolition of the release of MCP-1,will lead to an event where, theoretically there will be a recruitmentof neutrophils but not mononuclear cells. This hypothesis is inaccordance with the pathological state of gout and pseudogout, where thepredominant inflammatory cell is the neutrophil (Hachicha et aL, 1995).In addition MSU crystal activation of mononuclear phagocytes, which arenormally found in the joint space, also induces secretion of IL-8(Terkeltaub et al., 1991). The importance of IL-8 in this pathology hasbeen shown in synovial fluids of patients with acute gouty arthritiswhere it occurs in elevated amounts (Terkeltaub et al., 1991; di Giovineet al., 1991, J Clin. Invest. 87: 1375-1381). The use of a neutralisingantibody against IL-8 has been shown significantly to attenuate thecrystal induced joint swelling at 12 h and neutrophil infiltration intoarthritic joints at 12 and 24 h in a rabbit model (Nishimura et aL,1997, J. Leukoc. Biol. 62: 444-449).

[0295] These studies demonstrate the importance of both the emigratingneutrophil and the chemokine IL-8, as well as the release of this andother cytokines from resident cells such as the synoviocytes,macrophages and mast cells in treating gout. Since neutrophils are notpresent or are extremely rare in normal synovial fluid, enhancedneutrophil-endothelial adhesion is necessary for gout to occur(Terkeltaub, 1996, In. Koopman, W. J. editor. Arthritis and alliedconditions: a textbook of rheumatology. Baltimore: Williams and Wilkins:pp. 2085-2102, and Terkeltaub, 1992, In Inflammation. Basic Principlesand Clinical Correlates, ed. by J. I. Gallin, I. M. Goldstein and R.Snyderman, pp 977-981, Raven Press, New York). IL-1β and TNF-alpha maybe critical in mediating the rapid up-regulation of the majorendothelial ligand for neutrophils. For instance rapid and prolongedexpression of E-selectin in response to injection of urate crystals hasbeen demonstrated in pig skin (Chapman et al., 1996, Br. J Rheumatol.35: 323-334). The release of cytokines, chemokines and products of thearachidonic acid cascade system lead to the recruitment of neutrophilsin this pathology, and inhibition of these leads to an attenuation ofthe pathology.

[0296] The following gout model was used to test a PARP inhibitoraccording to the present invention.

[0297] Male outbread Swiss albino mice (20-22 g body weight) werepurchased from Banton and Kingsman (T. O. strain; Hull, Humberside) andmaintained on a standard chow pellet diet with tap water ad libitum anda 12:00 h light /dark cycle. All animals were housed for 1 week prior toexperimentation to allow body weight to reach 28-30 g.

[0298] 1,1 lb-dihydrobenzopyrano[4,3,2-de ]isoquinolin-1-one wasdissolved in 100% DMSO at room temperature at a concentration of 45 mgin 2 ml. The compound was then injected into the peritoneal cavity, soas each mouse received a single dose corresponding to 45 mg/2 ml/kg(e.g. 60 μl for a mouse of 30 g). Control mice received DMSO at 2 ml/kgi.p. A third group of mice which were left untreated were added tocontrol for potential effects of the vehicle. The study involvedtherefore, the following three groups: group A, untreated mice, n=6,group B, DMSO-treated mice, n=8, and group C, mice treated with 1,1lb-dihydrobenzopyrano[4,3,2-de ]isoquinolin-1-one, n−8

[0299] MSU crystal-induced neutrophil recruitment was tested as follows.In all cases, mice were treated 1 h after the treatment noted above,with MSU crystals. A homogenous suspension of MSU crystals was obtainedby a 30 min rotation. Peritonitis was induced by injection of 3 mg MSUcrystals in 0.5 ml PBS (0.1 M, pH 7.4), and the recruitment ofneutrophils into the cavity evaluated at the 6 h time point (Getting etal., 1997, J. Pharmacol. Exp. Ther. 283: 123-130). Animals were theneuthanised by CO₂ exposure and the peritoneal cavity washed with 3 ml ofPBS supplemented with 3 mM EDTA and 25 U/ml heparin.

[0300] An aliquot (100 μl) of the lavage fluid was then diluted 1:10 inTurk's solution (0.01% crystal violet in 3% acetic acid). The sampleswere then vortexed and 10 μl of the stained cell solution were placed ina Neubauer haematocymometer and neutrophils numbers counted using alight microscope (Olympus B061). Cell-free supernatants have beenprepared by centrifiugation and stored for potential future analysis.

[0301] Data are shown for single mice, and also shown as mean+S.E. of(n) mice per group. Statistical differences were determined by ANOVA,plus Bonferroni test. A P value<0.05 was taken as significant.

[0302] TABLE V reports the number of neutrophils as measured 6 hpost-MSU crystal injection in the three experimental groups. TABLE VEffect of 1,11b-dihydrobenzopyrano[4,3,2-de]isoquinolin-1-one on MSUcrystal induced neutrophil migration as evaluated at the 6 h time-point.Mouse Neutrophil No. Group Numbers 1 A 4.9 2 A 5.4 3 A 6.3 4 A 6.9 5 A5.7 6 A 6.0 7 8 1 B 6.0 2 B 6.6 3 B 7.5 4 B 7.8 5 B 5.1 6 B 5.7 7 B 5.78 B 6.0 1 C 5.1 2 C 2.1 3 C 2.4 4 C 2.4 5 C 3.0 6 C 3.0 7 C 2.7 8 C 2.1

[0303] TABLE VI illustrates these data as mean+S.E. It can be seen thatDMSO produced a modest not significant increase in cell migration (+7%).In contrast, the exemplary compound of the present invention, at thedose of 45 mg/kg, significantly reduced cell influx, with a calculated55% of inhibition vs. the vehicle group. TABLE VI Accumulation of datafor the effect of the exemplified compound of the present invention(means). Neutrophils Experimental Group Stimulus (10⁶ per mouse) A MSUcrystals (3 mg) 5.87 ± 0.28 (6) B MSU crystals (3 mg) 6.30 ± 0.33 (8) CMSU crystals (3 mg) 2.85 ± 0.34 (8) *

[0304] The invention being thus described, it will be obvious that thesame may be varied in many ways. Such variations are not to be regardedas a departure from the spirit and scope of the invention, and all suchmodifications are intended to be included within the scope of thefollowing claims. All references cited herein are incorporated in theirentirety by reference herein.

We claim:
 1. A compound of Formula I:

or a pharmaceutically acceptable salt, hydrate, prodrug, or mixturesthereof, wherein: R₁ hydrogen or halogen; R₂ is hydrogen, hydroxyl,amino, nitroso, methyl, amino methyl or carboxylic acid; one of R₃ andR₄ is —QP and the other of R₃ and R₄ is one of hydrogen, methyl,trifluoromethyl, nitro, amino, halogen and 1-piperazine, wherein Q isone of

P is Z—(R₅R₆)), Z or a 5 or 6 membered substituted or unsubstitutedaromatic or non-aromatic ring which contains 0, 1, 2 or 3 heteroatomsselected from the group consisting of O, N, S and a combination of twoor three of 0, N and S;. wherein A is carbon or S=O, X is 0, S , N or anN-substituted amino acid; provided that, when X is O or S, then Y isabsent, when X is N, then Y is hydrogen, C₁-C₆ straight or branchedchain alkyl, optionally substituted alkoxy or alkyl amino, or Y and Zare taken together to form a 5, 6 or 7 membered substituted orunsubstituted heterocyclic aromatic or non-aromatic ring which contains1, 2 or 3 heteroatoms selected from O, N, S and mixtures combination; Zis hydrogen, a direct bond, a carbonyl, an optionally substituted C₁-C₅straight or branched chain alkyl, cycloalkyl, carboxy, optionallysubstituted C₁-C₆ ether, aryl or heteroaryl, alkylalkenyl, alkynyl,alkylhalo, straight or branched chain or CH₂COOH, provided that when Pis Z, Z is not hydrogen and R₅ and R₆ are independently hydrogen, loweralkyl, lower alkenyl, lower alkanol, heterocycle, heteroaryl, alkoxy,aryloxy, alkylamino, arylamino, CH₂COOH, or R₅ and R₆ taken togetherform a 5, 6 or 7 membered substituted or unsubstituted heterocyclic ringcontaining 1, 2 or 3 heteroatoms selected from O, N, S and combinationsthereof.
 2. A compound of claim 1 selected from the group consisting of


3. A compound of claim 1 wherein R₂ is hydrogen, Q is

and X is N.
 4. A compound of claim 1 wherein R₂ is hydrogen, Q is

and X is N and A as S=O.
 5. A compound of claim 1 R₂ is hydrogen, Q is

and X is N.
 6. A pharmaceutical composition which comprises: (i) atherapeutically effective amount of a compound according to claim 1 and(ii) a pharmaceutically acceptable carrier.
 7. The pharmaceuticalcomposition of claim 6, wherein the carrier is a sterile solution,suspension, emulsion, a capsule or tablet.
 8. The pharmaceuticalcomposition of claim 6, wherein the carrier comprises a biodegradablepolymer or a solid implant.
 9. The pharmaceutical composition of claim 6for inhibiting PARP activity, treating or preventing diseases ordisorders, altering gene expression, or radiosensitizing.
 10. A methodof treating a disease or disorder selected from the group consisting oftissue damage resulting from cell damage or death due to necrosis orapoptosis, neuronal mediated tissue damage or diseases, neural tissuedamage resulting from ischenia and reperfusion injury, neurologicaldisorders and neurodegenerative diseases, vascular stroke,cardiovascular disorders, age-related muscular degeneration, AIDS andother immune senescence diseases, inflammation, arthritis, gout,atherosclerosis, cachexia, cancer, degenerative diseases of skeletalmuscle involving replicative senescence, hyperglycemia, diabetes, headtrauma, immune senescence, inflammatory bowel disorders, musculardystrophy, osteoarthritis, osteoporosis, chronic pain, acute pain,neuropathic pain, nervous insult, peripheral nerve injury, renalfailure, retinal ischiemia, septic shock, and skin aging, diseases ordisorders relating to lifespan or proliferative capacity of cells, anddiseases or disease conditions induced or exacerbated by cellularsenescence comprising administering a compound of claim 1 to an animalin need of said treatment.
 11. The method of claim 10 wherein theneurodegenerative disease is selected from the group consisting ofAlzheimer's Disease, Parkinson's Disease, Huntington's Disease andamyotropic lateral sclerosis.
 12. The method of claim 10, wherein thecancer is selected from the group consisting of ACTH-producing tumors,acute lymphocytic leukemia, acute nonlymphocytic leukemia, cancer of theadrenal cortex, bladder cancer, brain cancer, breast cancer, cervixcancer, chronic lymphocytic leukemia, chronic myelocytic leukemia,colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer,esophageal cancer, Ewing's sarcoma, gallbladder cancer, hairy cellleukemia, head & neck cancer, Hodgkin's lymphoma, Kaposi's sarcoma,kidney cancer, liver cancer, lung cancer (small and/or non-small cell),malignant peritoneal effusion, malignant pleural effusion, melanoma,mesothelioma, multiple myeloma, neuroblastoma, non-Hodgkin's lymphoma,osteosarcoma, ovary cancer, ovary (germ cell) cancer, prostate cancer,pancreatic cancer, penile cancer, retinoblastoma, skin cancer,soft-tissue sarcoma, squamous cell carcinomas, stomach cancer,testicular cancer, thyroid cancer, trophoblastic neoplasms, cancer ofthe uterus, vaginal cancer, cancer of the vulva and Wilm's tumor. 13.The method of claim 10 wherein said disease is gout.
 14. The method ofclaim 10, wherein the cardiovascular disorder is selected from the groupconsisting of cardiovascular tissue damage, coronary artery disease,myocardial infarction, angina pectoris and cardiogenic shock.